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Immobilizing Enzymes How to Create More SuitableBiocatalysts.

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Highlights
Immobilized Biocatalysts
Immobilizing Enzymes: How to Create More
Suitable Biocatalysts
Uwe T. Bornscheuer*
Keywords:
biotransformations · enzyme catalysis ·
immobilization · lipase · magnetic nanoparticles
Enzymes are versatile biocatalysts and
find increasing application in many
areas, including organic syntheses. The
major advantages of using enymes in
biocatalytic transformations are their
chemo-, regio-, and stereospecificity as
well as the mild reaction conditions that
can be used.[1] However, even when an
enzyme is identified as being useful for a
given reaction, its application is often
hampered by its lack of long-term stability under process conditions, and also
by difficulties in recovery and recycling.
These problems can be overcome by
immobilization, which provides the following advantages:
* enhanced stability,
* repeated or continuous use,
* easy separation from the reaction
mixture,
* possible modulation of the catalytic
properties,
* prevention of protein contamination
in the product,
* easier prevention of microbial contaminations.
Since the first uses of biocatalysts in
organic synthesis dating back almost a
century, researchers have tried to identify methods for linking an enzyme to a
carrier. Numerous examples of a broad
range of enzymes and reaction systems
(both aqueous systems and organic
solvents) have been documented in the
literature.[2] Although this reflects the
[*] Prof. Dr. U. T. Bornscheuer
Institut fr Chemie und Biochemie
Technische Chemie & Biotechnologie
Ernst-Moritz-Arndt-Universit(t Greifswald
Soldmannstrasse 16
17487 Greifswald (Germany)
Fax: (+ 49) 3834-86-4346
E-mail:
uwe.bornscheuer@uni-greifswald.de
3336
importance of biocatalysis, it also exemplifies that a general, broadly applicable
method for enzyme immobilization still
needs to be discovered. The most frequently used immobilization techniques
fall into four categories:
* noncovalent adsorption or deposition,
* covalent attachment,
* entrapment in a polymeric gel, membrane, or capsule,
* cross-linking of an enzyme.
All these approaches are a compromise between maintaining high catalytic
activity while achieving the advantages
of immobilization. Two recent trends
are based on 1) the use of new reagents
and/or carriers and 2) approaches that
take into account the increasing knowledge of enzyme structure and mechanism.
Reetz et al. described in 1995 that
the activity of lipases can be enhanced
up to 100-fold by immobilization in sol–
gels.[3] Cross-linked enzyme crystals
(CLECs)[4] were reported to give an
increase in enantioselectivity relative to
the native enzyme,[5] but this was mostly
attributed to the removal of a lessselective isoenzyme during CLEC preparation. Since crystallization of proteins
is not an easy task, cross-linked enzyme
aggregates (CLEA), which are obtained
by precipitation of proteins followed by
cross-linking with glutaraldehyde, might
represent an easy alternative. The
CLEA from pencillin acylase had the
same activity as a CLEC in the synthesis
of ampicillin, but the cross-linked aggregate also catalyzed the reaction in a
broad range of organic solvents.[6]
A promising combination of easy
separation and high stability has been
reported for a lipase immobilized on
magnetic g-Fe2O3 nanoparticles.[7] The
use of magnetic particles is not new,[8]
but Ulman and co-workers were able to
produce nanoparticles with an average
size of 20( 10) nm (instead of the usual
75–100 mm), which were then covalently
linked, after thiophene-functionalization, to a lipase from Candida rugosa.
The resulting biocatalyst exhibited significantly higher stability (over a period
of almost one month) than the native
enzyme in the hydrolysis of p-nitrophenyl butyrate (Figure 1). Moreover,
the immobilized enzyme can be easily
separated from the reaction mixture by
application of a magnetic field, which
either holds the immobilized enzyme in
place or removes it, as the nanoparticles
show very high magnetization values.
The increasing knowledge of enzyme structures and mechanism should
also enable more controlled immobilizations. For example, lipase from Pseudomonas fluorescens was immobilized
on four different carriers.[9] The native
enzyme and two carrier-linked lipase
preparations showed no or only modest
changes in activity and enantioselectivity in the kinetic resolution of a racemic
carboxylic acid ethyl ester. However,
two immobilizates exhibited substantially altered properties (Table 1). Specific
activity was increased sevenfold and
enantioselectivity increased from E = 7
to E = 86 for the lipase immobilized on
decaoctyl-sepharose. The authors claim
that, during this (also much more rapid)
immobilization procedure, the lipase
underwent a conformational change
from the closed to an open structure.
In this process, a hydrophobic “lid”,
which is known to be present in most
lipases, moves aside as a result of an
interfacial activation caused by the car-
DOI: 10.1002/anie.200301664
Angew. Chem. Int. Ed. 2003, 42, 3336 – 3337
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angewandte
Chemie
[2]
[3]
Figure 1. Stability of lipase from Candida rugosa immobilized on g-Fe2O3 nanoparticles during
the hydrolysis of p-nitrophenyl butyrate.[7] The concentration–time profiles (pNP = p-nitrophenol),
which were recorded between 1 (&) and 27 days (^, bottom curve), clearly shows the higher
stability of the immobilized lipase. Reproduced with permission from the American Chemical
Society.
[4]
Table 1: Comparison of different “controlled” immobilization techniques for a lipase from
Ps. fluorescens (PFL) in the resolution of a racemic carboxylic acid ester.[9]
Carrier
Activity [U mg 1][a]
Enantiomeric excess[b] [%]
Enantioselectivity
– (native)
dextran-agarose
glyoxyl-agarose
IHA-octyl-agarose[c]
IHA-decaoctyl-sepharose[c]
0.5
0.45
0.35
3.5
3.4
59
58
74
92
92.5
7
6.9
20
79
86
[5]
[6]
[7]
[a] mmol of substrate hydrolyzed per minute per mg of immobilized protein. [b] Enantiomeric
excess of the remaining (R)-ester. [c] IHA = interfacial hydrophobic adsorption.
[8]
rier and immobilization procedure and
provides enhanced access of the substrate to the active site residues. With a
similar strategy, the same research
group also reported the modulation of
the properties of penicillin acylases from
three different species, which also undergo conformational changes upon
binding of the acyl donor substrate.[10]
In the future, information derived
from protein sequences, 3D-structures,
and reaction mechanism should be further combined with the properties of
Angew. Chem. Int. Ed. 2003, 42, 3336 – 3337
carriers (functional groups, hydrophobicity, magnetic properties) and physical/chemical methods in order to produce a directed immobilization strategy.
[1] a) H. E. Schoemaker, D. Mink, M. Wubbolts, Science 2003, 299, 1694 – 1697;
b) A. Schmid, J. S. Dordick, B. Hauer,
A. Kiener, M. Wubbolts, B. Witholt,
Nature 2001, 409, 258 – 268; c) K. Faber,
Biotransformations in Organic Chemistry, 4th ed., Springer, Berlin, 2000;
d) U. T. Bornscheuer, R. J. Kazlauskas,
www.angewandte.org
[9]
[10]
Hydrolases in Organic Synthesis—Regio- and Stereoselective Biotransformations, Wiley-VCH, Weinheim, 1999.
a) T. Boller, C. Meier, S. Menzler, Org.
Process Res. Dev. 2002, 6, 509 – 519; b) J.
Lalonde, A. Margolin in Enzyme Catalysis in Organic Synthesis, Vol. 2 (Eds.: K.
Drauz, H. Waldmann), Wiley-VCH,
Weinheim, 2002, pp. 163 – 184; this review also contains a broad list of further
literature about enzyme immobilization.
a) M. Reetz, A. Zonta, J. Simpelkamp,
Angew. Chem. 1995, 107, 373 – 376;
Angew. Chem. Int. Ed. Engl. 1995, 34,
301 – 303; b) M. T. Reetz, P. Tielmann,
W. WiesenhHfer, W. KHnen, A. Zonta,
Adv. Synth. Catal. 2003, 345, 717 – 728.
a) N. Khalaf, C. P. Govardhan, J. J. Lalonde, R. A. Persichetti, Y. F. Wang,
A. L. Margolin, J. Am. Chem. Soc.
1996, 118, 5494 – 5495; b) T. Zelinski,
H. Waldmann, Angew. Chem. 1997, 109,
746 – 748; Angew. Chem. Int. Ed. Engl.
1997, 36, 722 – 724.
J. J. Lalonde, C. Govardhan, N. Khalaf,
A. G. Martinez, K. Visuri, A. L. Margolin, J. Am. Chem. Soc. 1995, 117, 6845 –
6852.
L. Cao, F. van Rantwijk, R. A. Sheldon,
Org. Lett. 2000, 2, 1361 – 1364.
A. Dyal, K. Loos, M. Noto, S. W. Chang,
C. Spagnoli, K. V. P. M. Shafi, A. Ulman,
M. Cowman, R. A. Gross, J. Am. Chem.
Soc. 2003, 125, 1684 – 1685.
a) L. Cao, U. T. Bornscheuer, R. D.
Schmid, J. Mol. Catal. B 1999, 6, 279 –
285; b) R. F. H. Dekker, Appl. Biochem.
Biotechnol. 1989, 22, 289 – 310.
G. FernKndez-Lafuente, M. Terreni, C.
Mateo, A. Bastida, R. FernKndez-Lafuente, P. Dalmases, J. Huguet, J. M.
Guisan, Enzyme Microb. Technol. 2001,
28, 389 – 396.
a) M. Terreni, G. Pagani, D. Ubiali, R.
FernKndez-Lafuente, C. Mateo, J. M.
Guisan, Bioorg. Med. Chem. Lett. 2001,
11, 2429 – 2432; b) S. Rocchietti, A. S. V.
Urrutia, M. Pregnolato, A. Tagliani,
J. M. Guisan, R. FernKndez-Lafuente,
M. Terreni, Enzyme Microb. Technol.
2002, 31, 88 – 93.
2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3337
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