Entrapment of lipases in hydrophobic sol-gel-materials: Efficient heterogeneous biocatalysts in aqueous medium

Article abstract of DOI:10.1055/s-2000-6276

The entrapment of certain lipases such as SP 525 (Candida antarctica B) or SP 523 (Humicola lanuginosa) in hydrophobic sol-gel materials derived from mixtures of RSi(OMe)₃ (R = alkyl) and Si(OMe)₄ or other alkyl-modified silane precu

Full text of DOI:10.1055/s-2000-6276

SHORT PAPER
781
Entrapment of Lipases in Hydrophobic Sol-Gel-Materials: Efficient
Heterogeneous Biocatalysts in Aqueous Medium
Manfred T. Reetz,*a Regina Wenkel,a David Avnir
a Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim/Ruhr, Germany
b Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Fax +49(208)3062985, E-mail: reetz@mpi-muelheim.mpg.de
Received 18 February 2000
matrix.4,5 One possible explanation of this "alkyl effect" is
a type of activation in the microenvironment of the en-
zyme based on the interaction of the alkyl groups with the
hydrophobic or amphiphilic domains of the lipase, akin to
Abstract: The entrapment of certain lipases such as SP 525 (Can-
dida antarctica B) or SP 523 (Humicola lanuginosa) in hydropho-
bic sol-gel materials derived from mixtures of RSi(OMe)3
(R = alkyl) and Si(OMe)4 or other alkyl-modified silane precursors
results in heterogeneous biocatalysts which are remarkably active in classical interfacial activation of lipases which is known
the hydrolysis of esters in aqueous medium. Relative activities of
to occur at the interface between water and droplets of li-
pophilic substrates such as triglycerides.1,7 The hydrolysis
50−100% are typical.
Key words: enzymes, catalysis, ester hydrolysis, sol-gel, heteroge-
neous catalysis
of esters is traditionally performed with lipases fully dis-
solved in water, allowing for maximum dispersion in this
medium. Thus, in hydrolysis, high enzyme activity of li-
pase-containing gels is not self-evident because these het-
erogeneous biocatalysts are completely insoluble. We
now report the unexpected observation that certain hydro-
phobic sol-gel materials containing entrapped lipases do
in fact display a remarkable degree of enzyme activity
when used as heterogeneous biocatalysts in hydrolysis re-
actions.
Lipases are the most used enzymes in synthetic organic
chemistry, catalyzing the chemo-, regio- and enantiose-
lective hydrolysis of carboxylic acid esters.1 When used
as a powder in organic solvents, the reverse reaction (es-
terification) as well as transesterification is possible.1,2
However, such processes are economically and ecologi-
cally viable only if the lipases can be immobilized effi-
ciently using cheap methods, and if such immobilizates
can be separated from the reaction products and used
again without significant loss of enzyme activity.3 We
have previously shown that lipases can be entrapped in
hydrophobic sol-gel materials resulting in heterogeneous
biocatalysts which display long-term stability as well as
pronounced degrees of enhanced activity in esterification
and transesterification reactions in organic solvents.4,5
The preparation of the catalysts involves the base-cata-
lyzed hydrolysis of silanes RSi(OMe)3 (R = alkyl) or mix-
tures of these or other silanes with tetramethoxysilane
Si(OMe)4 (TMOS) in the presence of a lipase (Scheme 1).
The hydrolysis of ester 1 was chosen as the test reaction
because one of the products 2 can easily be detected by
UV/Vis spectroscopy at 410 nm (Scheme 2). Since the es-
ter is not fully soluble in water, a small amount of acetone
was added (13%). In order to determine relative activities,
reactions using the gels and those involving solutions of
non-immobilized enzymes were compared using the same
amount of enzyme and solvents, in both series samples be-
ing periodically taken for UV/Vis-spectroscopic analysis
over the entire reaction. In the case of the gels, the samples
were first passed through a filter to remove the solid ma-
terial, which interferes with the UV-analysis. In all exper-
iments, the reaction vessels were vigorously shaken at the
same rate at room temperature. Satisfactory reproducibil-
ity with respect to enzyme activity was observed (± 15%).
H2O
RSi(OMe)3
lipase-immobilizate
lipase
O
Preparation of lipase containing gels
O
OH
Scheme 1
O
H2O
lipase-
immobilizate
+
OH
In esterification reactions such gels display relative activ-
ities of 500−8000% with respect to the traditional use of
the corresponding enzyme powders in organic solvents.4
Relative activity is defined as [ν(immobilized lipase)/
ν(commercial enzyme)]∑100%, where ν is the initial rate
of the reaction in each case. If TMOS is used alone,6 the
corresponding hydrophilic SiO2-gels lead to enzyme ac-
tivities of only 5%, demonstrating the necessity of incor-
porating hydrophobic alkyl groups in the silicon oxide
NO2
NO2
1
2
3
Scheme 2
In all cases the lipases were entrapped as previously de-
scribed,4 in the present case a low loading (about 1%) be-
ing chosen due to the relatively high inherent reactivity of
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782
M. T. Reetz et al.
SHORT PAPER
the
substrate. Compounds
Si(OMe)4
(TMOS), over, in control experiments involving the alkyl-modified
CH3Si(OMe)3 (MTMS), n-C3H7Si(OMe)3 (PTMS), hydrophobic gels in the absence of lipases, no catalytic
HO[Me2Si-O]nSi(Me)2OH (PDMS) or mixtures thereof activity was observed. Finally, although the kinetic results
were chosen as the sol-gel precursors. The results for a of the catalytically most active lipase-containing gels
number of different lipases and gels are summarized in refer to initial reaction rates, the conversion is in fact com-
Figure 1. Many of the entrapped lipases such as those plete within 20 minutes.
from Candida antarctica, Rhizopus arrhizus, etc. appear
How can an insoluble lipase-containing gel be almost or
to be unsuitable catalysts for the hydrolysis reaction, an
just as active as a homogeneous aqueous solution of the
observation that we originally expected of all lipase gels.
same enzyme? A simple explanation could be extensive
leaching into the aqueous phase. However, although small
amounts of leaching are difficult to exclude, this cannot be
the complete explanation in view of the results obtained in
In contrast, the entrapped lipases from AMANO PS and
Pseudomonas fluorescens display respectable relative ac-
tivities of 20−30%, provided hydrophobic matrices are
chosen. A related result was recently reported in the hy-
recycling experiments. For example, the model reaction
drolysis of certain carbohydrate esters, although in that
was performed eight times in succession using the PTMS/
study optimization of the sol-gel materials with respect to
TMOS-derived gel containing the entrapped lipase from
Candida antarctica B (SP 525). After each reaction the
gel was filtered off, washed successively with water and
acetone, and then reused, the residual activity in the model
the type of lipase was not performed.8 In our study, we
observed that the model reaction 1 → 2 + 3 is catalyzed
rather efficiently by the entrapped lipase SP 525 from
Candida antarctica B, the relative activity amounting to
reaction being measured by UV/Vis spectroscopy. As Fig-
52%. Encouraged by these unexpected results, we ex-
ure 2 reveals, in each run only about 5% enzyme activity
plored the use of other lipases, a search that led to the dis-
is lost. This could be due to mechanical loss, leaching, or
covery that certain sol-gel entrapped forms of the lipase
both. We have previously shown that mechanical stability
of the gels can be greatly enhanced if the sol-gel process
SP 523 (Humicola lanuginosa) are exceptionally active
heterogeneous catalysts. Relative enzyme activities of
is performed in the presence of porous glass beads such as
93%, 107%, and 110% were observed upon entrapping
SIRAN®, leading to the attachment of the enzyme-con-
this lipase in PDMS/TMOS (1:3), PDMS/MTMS (1:3),
taining gels in the giant pores of such solid supports.9 This
and PTMS/TMOS (5:1), respectively. These remarkable
was also accomplished in the present case. Figure 2 shows
observations show that enzyme activities in the homo-
that the activity of this heterogeneous biocatalyst in aque-
geneous and heterogeneous systems are actually compa-
ous medium remains constant over at least eight runs! In
rable. The same enzyme entrapped in traditional non-
striking contrast, the material prepared by adsorbing the
hydrophobic silica gel (SiO2)prepared from TMOS shows
lipase on SIRAN® itself loses its activity almost complete-
an activity of less than 7%, in line with the other observa-
ly after only two reactions, very likely due to strong leach-
tions concerning this particular matrix (Figure 1). More-
ing (Figure 2).
Figure 1 Relative activity of representative lipases entrapped in various sol-gel materials.
Synthesis 2000, No. 6, 781–783 ISSN 0039-7881 © Thieme Stuttgart · New York

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Entrapment of Lipases in Hydrophobic Sol-Gel-Materials
783
typical loading being 0.9%) at r.t. Shaking was continued at 300
revolutions/min, and samples were taken at regular intervals for
UV/Vis analysis.
Acknowledgement
We thank the German Israeli Foundation (GIF) for financial sup-
port, including a doctoral stipend for R. Wenkel.
References
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Figure 2 Residual activity of immobilized lipase SP 525 in the test
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In conclusion, we have discovered that some (but not all)
lipases entrapped in hydrophobic sol-gel materials are
surprisingly active heterogeneous catalysts for ester hy-
drolysis reactions in aqueous medium. Significantly, in
optimal cases it is possible to recycle the catalysts with lit-
tle or no decrease in enzyme activity. Aside from the prac-
tical implications regarding chemo-, regio- and
enantioselective hydrolytic reactions, our results show
that the previously reported "alkyl effect" observed in es-
terification and transesterification in organic solvents4,5
also operates in hydrolysis reactions performed in water.
Although a type of interfacial activation1,7 in the hydro-
phobic microenvironment of the solid matrix may be the
cause of this unusual phenomenon,5 further studies are
needed before final mechanistic conclusions can be made.
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Lipase-Containing Gel; Typical Procedure
Using the general procedure previously described,4b the mixture of
de-ionized water (364 µL), aq NaF-solution (1 M, 100 µL), an aq
PVA-solution (polyvinyl alcohol; mw 15000; 4% w/v, 200 µL) and
an aq lipase solution (200 µL, e.g., total of 5 mg of Candida antarc-
tica) is treated at r.t. successively with PTMS (875 µL, 5 mmol) and
TMOS (148 µL, 1 mmol). The two-phase mixture is vigorously
mixed for 3 s using a vortex mixer and then shaken by hand until the
evolution of heat sets in (1−2 min). The milky emulsion clears up
for a short time before solidifying rather rapidly with formation of
a white solid material. It is cooled at 0−5 °C for about 10 min and
aged at r.t. for 24 h. The material is dried at 37 °C for 3 d under nor-
mal pressure, then ground in a mortar and shaken with de-ionized
water (4 mL) for 2 h (350 revolutions/min). The product is collected
on a glass frit (D4), washed with de-ionized H2O (20 mL), acetone
(20 mL) and pentane (20 mL). The gel is then dried at 37 °C for 24
h and ground in a mortar. Note that due to the high activity of the
substrate in the model reaction, enzyme loading was chosen to be
low. For less reactive substrates loading should be increased by a
factor of 5−10. Indeed, in some commercially available lipase-con-
taining gels,10 loading is too low for any application.
van Tilbeurgh, H.; Egloff, M. -P.; Martinez, C.; Rugani, N.;
Verger R.; Cambillau, C. Nature (London) 1993, 362, 814.
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Brzozowski, A. M.; Derewenda, U.; Derewenda, Z. S.;
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(8) Pfau, R.; Kunz, H. Synlett 1999, 1817.
(9) Reetz, M. T.; Zonta; A.; Simpelkamp, J.; Könen, W. Chem.
Commun. (Cambridge) 1996, 1397.
(10) Some of the lipase-containing gels with low loading are
commercially available from Fluka (Switzerland).
Activity Test
A 50 mL Corning vessel equipped with a screw cap is charged with
Na-phosphate buffer (17.4 mL, 0.1 M, pH 7), p-nitrophenyl propi-
onate (1, 500 µL) (8 × 10−4 mM; 10 mg/mL of acetone) and acetone
(2 mL). At the start of the reaction an enzyme H2O-solution (100
µL) was added (or the corresponding amount of immobilized form,
Article Identifier:
1437-210X,E;2000,0,06,0781,0783,ftx,en;H01100SS.pdf
Synthesis 2000, No. 6, 781–783 ISSN 0039-7881 © Thieme Stuttgart · New York