Formation of silicones mediated by the sponge enzyme silicatein-α

Article abstract of DOI:10.1039/b921640e

The sponge-restricted enzyme silicatein-α catalyzes in vivo silica formation from monomeric silicon compounds from sea water (i.e. silicic acid) and plays the pivotal role during synthesis of the siliceous sponge spicules. Recombinant silicatein-α, which was cloned from the demosponge Suberites domuncula (phylum Porifera), is shown to catalyze in vitro condensation of alkoxy silanes during a phase transfer reaction at neutral pH and ambient temperature to yield silicones like the straight-chained polydimethylsiloxane (PDMS). The reported condensation reaction is considered to be the first description of an enzymatically enhanced organometallic condensation reaction. The Royal Society of Chemistry.

Full text of DOI:10.1039/b921640e

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Formation of silicones mediated by the sponge enzyme silicatein-a
Stephan E. Wolf,a Ute Schlossmacher,b Anna Pietuch,a Bernd Mathiasch,a Heinz-C. Schro¨der,b
Werner E. G. Mu¨ller*b and Wolfgang Tremel*a
Received 15th October 2009, Accepted 9th March 2010
DOI: 10.1039/b921640e
The sponge-restricted enzyme silicatein-a catalyzes in vivo silica formation from monomeric silicon
compounds from sea water (i.e. silicic acid) and plays the pivotal role during synthesis of the siliceous
sponge spicules. Recombinant silicatein-a, which was cloned from the demosponge Suberites
domuncula (phylum Porifera), is shown to catalyze in vitro condensation of alkoxy silanes during
a phase transfer reaction at neutral pH and ambient temperature to yield silicones like the
straight-chained polydimethylsiloxane (PDMS). The reported condensation reaction is considered
to be the first description of an enzymatically enhanced organometallic condensation reaction.
undergoes a transitory pentavalent silicon species involving a
Introduction
donating bond of the imidazole nitrogen of histidine.7,19 The
enzymatic parameters for silicatein were recently quantified and
are close to those which had have been determined previously for
the related hydrolytic reaction of cathepsin L.20,21
The use of biocatalysts, employed either as isolated enzymes
or whole microbial cells, offers a remarkable arsenal of highly
selective transformations for state-of-the-art synthetic organic
chemistry. Over the last two decades, this methodology has become
an indispensable tool for asymmetric synthesis, not only at the
academic level, but also on an industrial scale. Biotransformations
have been applied for the synthesis and hydrolysis of esters, amides
and nitriles, various reduction and oxidation reactions or carbon–
carbon bond-forming systems. In particular, the combination of
selectivity and mild reaction conditions offer promising perspec-
tives for industrial and pharmaceutical applications with a key for
environmentally friendly chemistry.1-5
Only a few living taxa, e.g. sponges, radiolarians, some algae
like diatoms and silicoflagellates, construct their skeleton from
silica.6-10 Sponges (phylum Porifera) show the singular ability to
synthesize their siliceous skeleton enzymatically,11-13 in contrast
to other organisms which deposit silica in a template-controlled
manner.14 Some of the responsible enzymes, silicatein-a, silicase
or silintaphin, were isolated and cloned from different siliceous
sponges (class Demospongiae), e.g. Tethya aurantium or Suberites
domuncula.11,12,15
Silicateins were shown to undergo post-translational modifica-
tions, primarily phosphorylation16 and to share high sequence-
similarity to the endopeptidase cathepsin L.11,12 Cathepsins occur
in lysosomes and cleave amide bonds near hydrophobic amino
acid residues in P2 and P3 positions.17 They belong to the group
of papain-like peptidases which are characterized by an active
centre consisting of the catalytic triade histidine (His), asparagine
(Asn) and cystein (Cys).13,18 The catalytic triade in silicateins differs
by only one amino acid from the one of cathepsin L; cystein is
replaced by serine (Ser).13 By site-directed mutagenesis, the Ser-
His-Asp triad was shown to be crucial for the hydrolytic activity
of the silicateins. It was further proposed that the silicon substrate
The enzymes involved in silica metabolism, in particular sili-
catein and silicase,22,23 have attracted increasing attention because
of their potential applications in the field of nano-biotechnology
and biomedicine. Silica-based materials are used in many high-
tech products including microelectronics, optoelectronics, and
catalysis. Biocatalysis of silica formation from water-soluble
precursors, in particular silicatein-mediated biosilica production,
occurs under mild physiological conditions and is advantageous
compared to technical (chemical) production methods which
require high temperatures, pressures or extremes of pH. In
addition, biological organisms such as sponges and diatoms are
able to fabricate their skeletons with high fidelity and in large
copy number. These properties are of extreme importance for
potential applications in nano-biotechnology. Silicatein remains
functionally active even after immobilization of the protein onto
metal or metal oxide surfaces.24
Silicatein is known to catalytically enhance the deposition of
silica and related metal oxides from a variety of precursors at
physiological temperature, pressure and neutral pH.25-28 Likewise,
recombinant silaffin, a silica-precipitating protein from the diatom
Cylindrotheca fusiformis, was repeatedly and successfully em-
ployed to synthesize aggregates or layers of titania nanoparticles
under ambient conditions from silicium(IV) resp. titanium(IV)
alkoxides.29,30 Actually, the R5 peptide, which is derived from
repeating subunit of the silaffin gen, shows as well the capability
of titania and silica synthesis at mild and ambient conditions,31-33
and involves in fact a self-assembling process.34
In this contribution we have utilized the catalytic activity of
recombinant silicatein-a for the in vitro formation of silicones like
the non-branched polydimethyl-siloxane (PDMS) under ambient
conditions.
aInstitute for Inorganic Chemistry, Johannes Gutenberg-University, Dues-
bergweg 10–14, 55099, Mainz, Germany. E-mail: tremel@mail.uni-
mainz.de; Fax: +49 6131 39-25605; Tel: +49 6131 39-25135
Experimental section
bInstitute for Physiological Chemistry (Applied Molecular Biology), Jo-
hannes Gutenberg-University, Duesbergweg 6, 55099, Mainz, Germany.
E-mail: wmueller@uni-mainz.de; Fax: +49 6131 39-25243; Tel: +49 6131
392 5910
Recombinant silicatein-a was cloned in Escherichia coli as de-
scribed previously.12 The enzyme was stored at a concentration
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of 100 mg mL-1 in 20 mmol L-1 MOPS [3-(N-morpholino)
propanesulfonic acid] buffer (pH 7.5, 50 mmol L-1 sodium acetate,
1 mmol L-1 EDTA). For control assays, heat-denatured silicatein
was used (95 ◦C for 15 min).
(s, 1, Si) ppm. (ether, saturated with water): -4.42 (s, 1, Si) ppm.
Methoxytetramethyldisilan-3-ole (dry ether): -11.79 (s, 1, Si–OH);
-11.97 (d, 1, Si–OMe) ppm. (ether, saturated with water): -11.08
(s, 1, Si–OH); -11.63 (s, 1, Si–OMe) ppm.
In situ UV/VIS characterization
Enzymatic preparation of silicones and their characterization
By substituting the p-aminophenoxy chromophoric group for the
methoxy group of the DMS substrate, it was possible to monitor
the cleavage and hydrolysis of the monomeric educt in situ. A
solution containing 40 mg of silicatein-a in 2.75 mL MOPS buffer
was covered with 0.75 mL of 2 mg mL-1 bis(p-aminophenoxy)-
dimethylsilane (APS, ABCR) in ether. The two-phase system was
stirred in Suprasil mixing cuvettes (Hellma QS-110) at 20 ◦C,
and the absorption of the aqueous phase was monitored between
220 and 800 nm (Varian Cary 5G UV/VIS spectrophotometer,
Mulgrave, Australia). Kinetic measurements were started 30 s after
addition of the components. Several controls were performed: (a)
in the absence of silicatein, (b) by replacing heat denaturated
silicatein for the active silicatein, or (c) by employing bovine
serum albumin (BSA, 50 mg in 2.75 mL MOPS buffer) instead
of silicatein. In addition, the reaction was performed by adding
water-soluble sodium hexafluorosilicate to the buffer solution to
compete with the APS substrate.
Silicatein-a (40 mg) dissolved in 2.5 mL MOPS buffer was covered
with 2.5 ml of dimethoxy dimethylsilane (DMS, ABCR) dissolved
in diethyl ether (p.a., Sigma-Aldrich, 10 : 1 v/v). Sampling was
done after incubation periods of 1 h, 3 h, and 5 h at 20 ◦C
under intense shaking (200 rpm, Promax 1020, Heidolph). In
control assays, heat-denaturated silicatein or bovine serum al-
bumine (BSA, Sigma-Aldrich) or no protein was added. Fur-
ther experiments were performed with educt mixtures of DMS
with trimethoxymethylsilane (TMMS, supplied by ABCR) or
trimethoxyphenylsilane (PTMS, supplied by ABCR) at molar
different ratios (1 : 0, 0 : 1, 1 : 4, 4 : 1, 1 : 9). The aqueous layer
which contained water-soluble decomposition products, silicatein
and buffer, was removed. Prior to investigation, the organic phase
containing the substrate (e.g. DMS) and silicone condensates (e.g.
PDMS), was dried with sodium sulfate (anhydrous, p.a., Sigma-
Aldrich) to prevent further decomposition. The condensation
products were characterized by EI mass spectrometry (Finnigan
MAT mass spectrometer 8230, Midland, Canada) and 29Si DEPT
NMR (DRX400, Bruker Biospin, Rheinstetten).
Results
A comparison of the MALDI-MS spectra of PDMS obtained in
presence and in absence of active silicatein shows a distinct increase
of the product chain length. In the absence of silicatein, oligomers
with up to seven repeat units were detected, whereas in the presence
of active silicatein the highest mass signal could be assigned to a
oligomer consisting of twelve monomer units (Fig. 1).
29Si DEPT NMR
29Si DEPT NMR measurements were performed in non-
deuterated ether locked on the proton signal of an external D2O-
filled capillary and calibrated to tetramethylsilane. The data was
recorded with a digital NMR spectrometer, (Advance DRX400,
Bruker Biospin) operating at 9.4 T and 79.5 MHz in case of 29Si. A
typical run consisted of ca. 300 DEPT scans with J = 6.6 Hz and
2 s of relaxation time, which is efficient to characterize –SiCHx,
–SiOCHx and even –29Si–SiCHx. The assignment of the 29Si NMR
signals of dimethyl silandiole and tetramethyldisilan-3-ole was
validated by comparison with standards synthesized according to
standard protocols.35 The corresponding mixed hydrolysis prod-
ucts dimethyl methoxysilanole and 1-methoxy tetramethyldisilan-
3-ole were prepared by quenching an analogous reaction with one
equivalent of methanol.
NMR signals of the organic phase originating from two-phase
reactions showed a slight shift to lower chemical displacements,
which is attributed to traces of water dissolved in the organic
phase because 29Si signals of silanols are known to shift with
increasing donor ability of the respective solvent.36 NMR spectra
of synthesized silicon standards recorded in ether saturated with
water approved that the observed shift to lower chemical displace-
ment is due to a trace of dissolved water. The employed water-
saturated ether (approx. 1.8% v/v) was prepared by vigorously
shaking diethyl ether (p.a., Sigma-Aldrich) against ultrapure water
(Millipore Synergy 185 with UV photo oxidation, 18.2 MX/cm)
for several days.
29Si NMR analysis showed silicatein-a to enhance the monomer
cleavage and the subsequent condensation to silicones consid-
erably. In the absence of silicatein, the DMS monomer was
hydrolyzed only to a negligible extent. Signals of two hy-
drolysis intermediates, dimethyl methoxysilanole and 1-methoxy
tetramethyldisilan-3-ole, could be identified. A low constant signal
ratio of 0.05 of the decomposition products to the monomer
during 5 h of incubation (Fig. 2a) indicated the stability of the
starting compound. In contrast, in the presence of silicatein-a the
NMR spectrum changed significantly with time (Fig. 2b), and new
signals of silandiols (dimethyl silandiole and tetramethyldisilan-
3-ole) appeared after 1 h of incubation. The signal ratio of
the hydrolysis products to the monomeric starting compound
increased drastically to 0.22 after 1 h, 0.89 after 3 h, and
2.31 after 5 h of incubation with active silicatein. In particular,
the NMR spectra of the sample incubated for 5 h showed
obvious changes (Fig. 3) having tetramethyldisilan-3-ole as the
predominant component. In control assays with denaturated
silicatein and without silicatein, only traces of the hydrolysis
intermediate methoxytetramethyldisilan-3-ole were found. These
results demonstrate the catalytic activity of silicatein-a in the
cleavage of the alkoxy bonds in alkoxy silanes.
Dimethylsilandiole (dry ether): -7.73 (s, 1, Si) ppm. (ether,
saturated with water): -6.62 (s, 1, Si) ppm. Tetramethyldisilan-3-
ole (dry ether): -14.92 (s, 2, Si) ppm. (ether, saturated with water):
-14.62 (s, 1, Si) ppm. Dimethylmethoxysilanole (dry ether): -5.21
Higher oligomers (n > 2) could not be detected by 29Si NMR.
Typically, species can only be detected by NMR techniques if
their concentration exceeds approx. 1 mol%. But the presence
of higher oligomers was demonstrated by mass spectrometry
9246 | Dalton Trans., 2010, 39, 9245–9249
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Fig. 1 EI mass spectrum of siloxane polymers. The mass distributions
of PDMS obtained in the presence of silicatein (b) differ significantly
from the distribution obtained in a control reaction within silicatein (a). A
distinct increase in the chain length of the product was observed in contrast
to PDMS reaction that proceeded in the absence of silicatein. The mass
difference of ~75 Da between the individual peaks corresponds to a single
[Si(Me)2O] unit.
Fig. 2 29Si NMR spectrum of PDMS formed from the monomer DMS
after 1, 3 and 5 h. The reaction was performed (a) in the absence of
silicatein or (b) in the presence of the enzyme. All spectra are normalized
with respect to their most intense signals.
(vide supra). The amount of higher oligomers decreased signifi-
cantly with chain length, and therefore reached the detection limit
of the NMR technique (Fig. 1).
In additional presence of varying amounts of either TMMS or
PTMS, the silicatein-enhanced polymerization of DMS is com-
petitively inhibited. The phenylated substrate PTMS blocked the
polymerization reaction completely as traceable in the detection
limit of the 29Si NMR technique. This can be rationalized by
both (a) steric hindering due to the bulky phenoxy group and
(b) by a decreased reactivity of the substrate to hydrolyze. The
phenoxy residue increases electron density at the silicon centre
of the substrate PTMS and thus decreases the affinity for a
nucleophilic attack of silicatein at the central silicon atom of the
substrate.
Replacing the methoxy group by the p-aminophenoxy chro-
mophor allowed an in situ monitoring of the reaction by UV/VIS
absorption spectroscopy. Fig. 4a shows the evolution of the
absorption spectra with time by the increasing concentration of the
decomposition product p-aminophenol. The plot of absorption
at 290 nm versus time showed sigmoidal characteristics of the
decomposition of the APS substrate (Fig. 4b), which allowed
recently the extraction of the Michaelis constant Km = 22.7 mM.21
In the presence of silicatein-a, the increase in the absorption
was accelerated considerably compared to control assays with
BSA or without protein. Saturation, which we defined as 98%
of the upper asymptote of a Boltzmann fit, was reached after
16 min of incubation with silicatein-a, whereas in control assays
saturation was reached only after 26 min. The reaction was
strongly competitively suppressed by adding the water-soluble
Fig. 3 29Si NMR spectrum of PDMS formed in the presence of silicatein
after 5 h of incubation with signal assignments.
substrate ammonium hexafluorosilicate to the aqueous layer. For
heat-denatured silicatein a decrease of the conversion rate (Fig. 4b)
was observed, even in comparison to control assays in the absence
of protein or in the presence of BSA, respectively. Saturation
was reached not until 50 min. From this we conclude that
denaturated silicatein exhibits a surface activity which can block
the phase-transfer of APS from the ether to the aqueous phase;
this leads to the observed decrease in the conversion rate. One
may speculate from this that heat-denatured silicatein-a exposes
hydrophic regions and thus have an increased tensioactivity.
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©

Silicateins, which are exclusively found in sponges, catalyse
in vivo silica formation from monomeric silicic acid esters. The
enzymatic activity of recombinant silicatein was studied in vitro for
transformation of alkoxy silanes to silicones (siloxanes) in a phase-
transfer reaction at neutral pH and under physiological conditions.
An extensive increase in the formation rate of oligomers and
in the chain length of the silicones could be demonstrated by
mass spectrometry, UV/Vis and NMR techniques. To the best
of our knowledge, the formation of silicones in the presence of
silicatein represents the first enzymatically driven organometallic
condensation reaction. UV/Vis-measurements and 29Si DEPT
NMR were used as a analytical probe to determine the enzymatic
activity of silicatein-a and revealed the hydrolytic activity of
silicatein-a.
The extraordinary feature of silicatein to produce silica bio-
catalytically under physiological conditions (neutral pH, ambient
temperature and pressure) can be utilized for the synthesis of
silicones. Therefore this biocatalytical reaction may have an impact
for the sustainable synthesis of silicones.43
Acknowledgements
We are grateful to the Deutsche Forschungsgemeinschaft (DFG)
for support within the priority program 1420 Biomimetic Materials
Research: Functionality by Hierarchical Structuring of Materials.
Stephan E. Wolf is a recipient of a fellowship from the Konrad
Adenauer-Foundation. In addition we acknowledge partial sup-
port from the European Commission, the Bundesministerium fu¨r
Bildung und Forschung Germany (Center of Excellence project
BIOTECmarin), and the International Human Frontier Science
Program.
Fig. 4 a) Absorption spectrum of the aqueous layer during phase-transfer
reaction in absence of silicatein. The absorption increases due to the
increasing concentration of the decomposition product p-aminophenol.
b) Time dependence of the absorption at 290 nm. The reactions were
performed in the presence of silicatein-a (᭹), heat-denaturated silicatein
(ꢀ), BSA (᭺) and in the absence of protein (ꢀ). The increase in absorption
shows a sigmoidal behaviour. In the conducted phase-transfer reaction,
the amount of available substrate should remain at a high level and thus
the reaction may cease by reason of an inactivation of the enzyme. The
smaller absorption for inactive silicatein can be related to a blocking of the
substrate phase-transfer due to tensioactivity of heat-denatured silicatein.
The reaction kinetics determined in assays with BSA (᭺) are comparable
to those recorded in the control assays without silicatein.
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