Recombinant silicateins as model biocatalysts in organosiloxane chemistry

Article abstract of DOI:10.1073/pnas.1613320114

The family of silicatein enzymes from marine sponges (phylum Porifera) is unique in nature for catalyzing the formation of inorganic silica structures, which the organisms incorporate into their skeleton. However, the synthesis of organosiloxanes catalyzed by these enzymes has thus far remained largely unexplored. To investigate the reactivity of these enzymes in relation to this important class of compounds, their catalysis of Si-O bond hydrolysis and condensation was investigated with a range of model organosilanols and silyl ethers. The enzymes' kinetic parameters were obtained by a high-throughput colorimetric assay based on the hydrolysis of 4-nitrophenyl silyl ethers. These assays showed unambiguous catalysis with kcat/Km values on the order of 2-50 min-1 μM-1. Condensation reactions were also demonstrated by the generation of silyl ethers from their corresponding silanols and alcohols. Notably, when presented with a substrate bearing both aliphatic and aromatic hydroxy groups the enzyme preferentially silylates the latter group, in clear contrast to nonenzymatic silylations. Furthermore, the silicateins are able to catalyze transetherifications, where the silyl group from one silyl ether may be transferred to a recipient alcohol. Despite close sequence homology to the protease cathepsin L, the silicateins seem to exhibit no significant protease or esterase activity when tested against analogous substrates. Overall, these results suggest the silicateins are promising candidates for future elaboration into efficient and selective biocatalysts for organosiloxane chemistry.

Full text of DOI:10.1073/pnas.1613320114

Recombinant silicateins as model biocatalysts in
organosiloxane chemistry
S. Yasin Tabatabaei Dakhilia,b, Stephanie A. Caslina,b, Abayomi S. Faponlea,c, Peter Quayleb, Sam P. de Vissera,c
and Lu Shin Wonga,b,1
,
aManchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, United Kingdom; bSchool of Chemistry, University of Manchester,
Manchester M13 9PL, United Kingdom; and cSchool of Chemical Engineering and Analytical Science, University of Manchester, Manchester M13 9PL,
United Kingdom
Edited by Galen D. Stucky, University of California, Santa Barbara, CA, and approved May 24, 2017 (received for review August 10, 2016)
The family of silicatein enzymes from marine sponges (phylum
Porifera) is unique in nature for catalyzing the formation of
inorganic silica structures, which the organisms incorporate into
their skeleton. However, the synthesis of organosiloxanes cata-
lyzed by these enzymes has thus far remained largely unexplored.
To investigate the reactivity of these enzymes in relation to this
important class of compounds, their catalysis of Si–O bond hydro-
lysis and condensation was investigated with a range of model
organosilanols and silyl ethers. The enzymes’ kinetic parameters
were obtained by a high-throughput colorimetric assay based on
the hydrolysis of 4-nitrophenyl silyl ethers. These assays showed
nosiloxanes. Several attempts have been made to use hydrolytic
enzymes such as lipases and proteases for the hydrolysis and
condensation of the Si–O bond (11–13). Although they clearly
demonstrate the feasibility of the general concept, the range of
enzymes tested have so far met with only limited success in
regard to synthetic yield and substrate scope.
In contrast, poriferans (marine sponges) that use silica as part
of their inorganic skeleton use a family of enzymes termed the
silicateins to catalyze the polymerization of soluble silicates into
silica (14–16). The primary sequences of these enzymes have
been reported and they bear a remarkable homology with pro-
teases of the cathepsin family, with ∼65% sequence similarities
and ∼50% sequence identities relative to cathepsin L. Both en-
zymes share a similar Xaa–His–Asn catalytic triad at their active
site, although in the silicateins a Ser residue occupies the Xaa
position rather than Cys in cathepsin L. Previous reports have
shown that silicatein-α (Silα), the prototypical member of this
family, can catalyze the hydrolysis of ethoxysilanes such as tet-
raethoxysilane (TEOS) and triethoxyphenylsilane (17).
Because the silicateins have evolved specifically to manipulate
the Si–O bond, these enzymes may offer a better starting point
for further elaboration into practical biocatalysts in organo-
siloxane chemistry. This paper outlines the performance of het-
erologously produced Silα for both the hydrolysis and condensation
of a range of model organosiloxanes. In the process, the devel-
opment of a colorimetric high-throughput screening method
for silyl ether bond hydrolysis based on the 4-nitrophenoxylate
unambiguous catalysis with
kcat/Km values on the order of
2–50 min−1 μM−1. Condensation reactions were also demonstrated
by the generation of silyl ethers from their corresponding silanols
and alcohols. Notably, when presented with a substrate bearing
both aliphatic and aromatic hydroxy groups the enzyme preferen-
tially silylates the latter group, in clear contrast to nonenzymatic
silylations. Furthermore, the silicateins are able to catalyze trans-
etherifications, where the silyl group from one silyl ether may be
transferred to a recipient alcohol. Despite close sequence homol-
ogy to the protease cathepsin L, the silicateins seem to exhibit no
significant protease or esterase activity when tested against anal-
ogous substrates. Overall, these results suggest the silicateins are
promising candidates for future elaboration into efficient and se-
lective biocatalysts for organosiloxane chemistry.
silicatein biocatalysis organosilicon organosiloxane silyl ether
|
|
|
|
he organosiloxanes, compounds containing C–Si–O moieties,
Significance
T
represent a class of compounds with a truly diverse range of
applications. They are commonly used in the form of poly-
siloxane “silicone” polymers as components of industrial and
consumer products for a variety of purposes such as bulking
agents, separation media, protective coatings, lubricants, emul-
sifiers, and adhesives (1–3). Their use as auxiliaries in the
chemical synthesis of complex molecules is also long-established
(4–6). However, the production and synthetic manipulation of
these compounds are almost entirely dependent on chlorosilane
feedstocks, which are environmentally undesirable and energy-
intensive to produce (7, 8). Furthermore, organosiloxanes, which
are entirely anthropogenic in origin, are now known to be per-
sistent environmental contaminants because little attempt is
made to recover and recycle them (3). Synthetic routes that use,
and ultimately recycle, siloxanes and silanols as alternatives
would in principle be more environmentally sound.
Organosiloxanes are components in a huge variety of con-
sumer products and play a major role in the synthesis of fine
chemicals. However, their synthetic manipulation primarily
relies on the use of chlorosilanes, which are energy-intensive to
produce and environmentally undesirable. Synthetic routes
that operate under ambient conditions and circumvent the
need for chlorinated feedstocks would therefore offer a more
sustainable route for producing this class of compounds. Here,
a systematic survey is reported for the silicatein enzyme, which
is able to catalyze the hydrolysis, condensation, and exchange
of the silicon–oxygen bond in a variety of organosiloxanes
under environmentally benign conditions. These results sug-
gest that silicatein is a promising candidate for development of
selective and efficient biocatalysts for organosiloxane chemistry.
One possible strategy toward improved sustainability is to
harness enzymes for chemical processing. Such biocatalysts are
attractive because they offer highly efficient synthesis in terms of
yields and regio- and stereospecificity, together with an ability to
promote reactions under mild conditions and a minimal reliance
on halogenated or metallic feedstocks (9, 10). The use of en-
zymes to manipulate the Si–O bond would therefore potentially
offer more sustainable routes to the synthesis of many com-
pounds, as well as for the eventual recycling and reuse of orga-
Author contributions: S.Y.T.D., S.P.d.V., and L.S.W. designed research; S.Y.T.D., S.A.C., and
A.S.F. performed research; S.Y.T.D., S.A.C., A.S.F., P.Q., S.P.d.V., and L.S.W. analyzed data;
and S.Y.T.D., S.A.C., P.Q., S.P.d.V., and L.S.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. Email: l.s.wong@manchester.ac.uk.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1613320114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1613320114
PNAS
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16000
12000
8000
4000
0
chromophore is also reported. Additionally, the protease and es-
terase activity of Silα against analogous substrates is described.
Results and Discussion
Production and Characterization of Silα. To acquire this enzyme, a
synthetic vector containing cDNA encoding for the mature wild-
type Silα from Suberites domencula, fused to an N-terminal
hexahistidine tag and codon optimized for expression in Escherichia
coli, was used. It is known that the mature form of the protein is
highly hydrophobic and difficult to produce in soluble form (16, 18).
Thus, in attempts to improve its solubility the gene was also subcl-
oned with the sequences for a number of proteins known to enhance
solubility and folding. Sequences encoding for GST, thioredoxin,
small ubiquitin-like modifier, maltose binding protein, or trigger
factor (TF) were inserted between the hexahistidine tag and Silα
and the genes transformed into a variety of E. coli BL21(DE3)
strains including Arctic-Express and Origami. Expression trials
were then carried out by varying the induction conditions, includ-
ing the concentration of the induction agent (isopropyl β-D-1-
thiogalactopyranoside), incubation temperature, and incubation time.
Overall, these optimization experiments showed that all of the
candidate proteins expressed well in E. coli but only the TF-Silα
fusion gave the protein in soluble form. As expected, the Silα
(without any fusion tag) was almost entirely insoluble. However,
it was found that addition of the nondenaturing detergents Tri-
ton X-100 and CHAPS into the lysis buffer enabled the recovery
of sufficient levels of the soluble protein for some further studies.
Both the TF-Silα fusion and the solubilized wild-type Silα were
then purified to homogeneity (Fig. 1).
The isolated TF-Silα, and a Ser→Ala mutant at position 26
(Ser26Ala) of this protein produced using the same procedures,
were fully soluble and could be handled without any special
precautions. Size-exclusion chromatography of both these pro-
teins in isolated form showed a single well-defined species (SI
Appendix, Fig. S1A). The CD spectrum of each protein showed
clear secondary structural features, indicating the proteins were
not denatured or disordered (Fig. 2 and SI Appendix, Fig. S2).
The spectra of the mutant and unmodified protein essentially
overlap, showing that the mutation did not affect its overall
folding. The relative proportions of secondary structures were
also calculated from these spectra and were found to be within
4% of the values calculated from combining the crystallograph-
ically derived data of a cathepsin-silicatein chimera (18) and TF
(SI Appendix, Tables S1–S3).
TF-Silα
TF-Silα (Mutant-Ser26Ala)
-4000
-8000
-12000
190
210
230
250
Wavelength (nm)
Fig. 2. CD spectra plots of molar ellipticity against wavelength for TF-Silα
and TF-Silα(Ser26Ala).
In contrast, Silα could be maintained in a homogeneous state
only at dilute concentrations (micromolar range), which were
insufficient for CD measurements. However, analysis by non-
denaturing gel electrophoresis for Silα clearly showed a single
band, indicating nonaggregation (SI Appendix, Fig. S1B). The
protein advanced through the gel at a similar rate as the 25 kDa
reference protein, suggesting that it was of approximately similar
size, globular, and monomeric.
Determination of Enzymatic Activity. To confirm that the two
candidate proteins (Silα and TF-Silα) were catalytically compe-
tent, their hydrolytic activity against TEOS was tested and the
amount of precipitated silica quantified by the previously
reported silicomolybdic acid assay (17). The amount of silica
produced upon exposure of silicic acid (derived from acid hy-
drolysis of TEOS) to these enzymes was also measured. Both
assays demonstrated that the enzymes were active for both the
hydrolysis and subsequent condensation of silicate Si–O bonds
(SI Appendix, Fig. S3). In comparison, the heat-denatured en-
zyme and chymotrypsin (as a representative serine protease)
displayed only a small amount of nonspecific activity, likely due
to general hydrophobic interactions and nonspecific basic ca-
talysis (19). These results were in agreement with previous re-
ports using Silα derived from the native organisms (15, 17) and
tests with trypsin and papain that are known to reject TEOS as a
substrate (11, 17).
High-Throughput Colorimetric Assays for Silyl Ether Hydrolysis. To
investigate the scope of these recombinant silicateins in the
manipulation of a wider range of siloxanes, it was necessary to
develop a new screening methodology for this class of com-
pounds, because the silicomolybdic acid assay is incompatible
with organosilanes (compounds with C–Si bonds). For this pur-
pose, a series of 4-nitrophenoxy silyl ethers 1–3 was synthesized
for use as model substrates (Scheme 1). Here, it was envisaged
that hydrolysis of the Si–O bond would result in the release
of the corresponding silanols 4–6 and the strongly absorbing
nitrophenoxylate ion, which can be quantified by UV-visible
(UV-Vis) spectrophotometry.
49.6 kDa
23.1 kDa
Silα
23.1 kDa
-C
A
B
N-
-C
N-
Trigger Factor
Silα
His6
His6
Amount of protein (mg)
Amount of protein (mg)
These substrates were used to perform time-course experi-
ments with the two enzyme candidates (Fig. 3 and SI Appendix,
Figs. S4 and S5) and the data were further verified by GC-MS
analysis. It was further observed that the assay in fact generated a
yellow color that is visually observable (SI Appendix, Fig. S6),
thus making it fully colorimetric rather than only spectrometric.
These assays showed the 4-nitrophenoxy substrates are some-
what susceptible to hydrolysis, and even in the absence of any
catalyst an appreciable rate of product formation is detectable.
Nevertheless, assays that used the fully competent enzyme always
gave enhanced rates of hydrolysis compared with the control
experiments or the uncatalyzed reaction.
70 kDa
25 kDa
70 kDa
25 kDa
Fig. 1. Images of SDS/PAGE gels for Silα (A) and TF-Silα (B) after purification,
over a range of dilutions, demonstrating the homogeneity of the isolated
proteins. Also included above each gel image are the schematic represen-
tations of the protein constructs.
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Tabatabaei Dakhili et al.

enzyme could be invoked, although how this effect is related to
the pH is unclear at present.
Kinetic Analysis of TF-Silα and Silα-Catalyzed Silyl Ether Hydrolysis.
To further characterize this enzyme-catalyzed hydrolysis, a series
of assays were conducted to extract the Michaelis–Menten kcat
and Km values (Table 1 and SI Appendix, Figs. S7–S9). Based on
the pH study above, these assays were performed at pH 8.5 as an
acceptable compromise between relatively good enzyme activity,
low levels of background (uncatalyzed) hydrolysis, and avoidance
of substrate inhibition.
In general, the binding of both candidate proteins to all of the
substrates was relatively weak, with Km values in the micromolar
range. However, it did not follow in the expected trend of de-
creasing Km with increasing substrate steric bulk. The TDS silyl
ether 2 gave a lower Km than the smaller tert-butyldimethylsilyl
(TBDMS) analog across both TF-Silα and Silα. This observation
suggests that the enzymes display some selectivity in regard to
substrate shape that is not simply a function of overall steric bulk
of the substrate. The kcat values also followed this general trend,
with substrate 2 giving the lowest turnover.
Substrate
Abbreviationa
TBDMS-ONp
TDS-ONp
R1
Me
Me
iPr
R2
Me
Me
iPr
R3
tBu
Thxa
iPr
Product
1
2
3
4
5
6
TIPS-ONp
Scheme 1. Hydrolysis of model 4-nitrophenoxy silyl ethers. aNp, 4-nitrophenyl;
TBDMS, tert-butyldimethylsilyl; TDS, thexyldimethylsilyl; Thx, thexyl TIPS,
tri(iso-propyl)silyl.
Taken together, these results demonstrate the feasibility of
using 4-nitrophenoxy silyl ethers as a high-throughput assay and
show that both enzymes accelerated silyl ether bond hydrolysis
significantly above that of background uncatalyzed hydrolysis.
The overall catalytic efficiency (kcat/Km) did follow a trend of
falling with increasing bulk of the substrates. These values were
comparable with the data from cathepsin L against its standard
Cbz-Phe-Arg-NHNp dipeptide nitrophenylanilide substrate 7
(25), suggesting the kinetics data obtained for the silicateins
were plausible.
pH Dependence of TF-Silα Hydrolytic Activity. To investigate the
effect of pH on enzymatic silyl ether hydrolysis the initial rates of
hydrolysis of 1 catalyzed by TF-Silα over pH 6.5–10.5 were de-
termined. Here, the initial rates of reaction were used as a
measure of activity and the net enzyme-catalyzed rate was cal-
culated after accounting for the background nonenzymatic hy-
drolysis. It was found that at low substrate concentrations
(≤0.05 mM) higher activities were recorded in the basic pH
range with a maximum at pH ∼10 (Fig. 4A), consistent to the
optimum pH range of “alkaline” serine proteases (20–22). This
optimum is related to the ionization state of catalytic His resi-
due, which must be unprotonated for activity, and the concen-
tration of hydroxide ions that participate in the hydrolysis of the
acyl-enzyme intermediate (22).
Substrate inhibition was observed at lower pH values with this
effect evident at substrate concentrations above ∼0.06 mM and
∼0.10 mM at pH 6.5 and 7.5, respectively (Fig. 4B). This phe-
nomenon of substrate inhibition, though not frequently dis-
cussed, is known in many enzyme systems through a variety of
mechanisms (23, 24). Of the proposed mechanisms, the classical
substrate inhibition model where the substrate acts as an allo-
steric regulator is unlikely in this case because 1 bears little re-
semblance to silicic acid, the presumed natural substrate, and
there are no known compounds bearing C–Si bonds of biological
origin (3). Models where the increasing amounts of substrate
result in the formation of noncatalytic conformations of the
Ser26Ala Mutation at the TF-Silα Active Site. To confirm that the
catalysis did indeed involve the serine residue at the active site,
the Ser26Ala mutant of TF-Silα was then tested. Using TEOS as
a substrate, only a very small amount of silica was formed,
comparable to that of the heat-denatured enzyme and chymo-
trypsin (SI Appendix, Fig. S3A). These data are in agreement
with a previous report where the equivalent mutation in wild-
type Silα from Tethya aurantia resulted in the abolition of specific
catalysis (26). When tested against the chromogenic substrate 2 a
similar result was obtained, with the unmodified TF-Silα result-
ing in a clear positive result whereas only low activity was found
in the mutant, comparable to the other control experiments (Fig.
5A). Although greatly reduced in all cases, total loss of catalysis
is never observed due to residual nonspecific catalysis, as
previously noted.
Molecular Dynamics Modeling of Silα Binding with TBDMS-ONp. It
was notable that the enzymes were able to process large non-
polar organosiloxanes, albeit at a low rate. There is currently no
experimentally derived structure for any of the silicateins, but
previous models (27) have suggested that the active site pocket
TF-Silα
TF-Silα
Silα
TF-Silα
A
B
C
Silα
Silα
TF-Silα (Mutant-Ser26Ala)
No Enzyme
TF-Silα (Mutant-Ser26Ala)
No Enzyme
TF-Silα (Mutant-Ser26Ala)
No Enzyme
10
8
20
15
10
5
35
30
25
20
15
10
5
6
4
2
0
0
0
0
200 400 600 800 1000
Time (min)
0
300 600 900 1200
Time (min)
0
50
100
150
Time (min)
Fig. 3. Graphs of the extent of hydrolysis of the 4-nitrophenoxy silyl ether substrates 1 (A), 2 (B), and 3 (C) against time, as measured by UV-Vis absorbance at
405 nm corresponding to the 4-nitrophenoxylate anion (calibrated using data from SI Appendix, Fig. S4). Assays are conducted with 0.00067 mol eq of enzyme
relative to 0.1 mM substrate in 5% 1,4-dioxane, 50 mM Tris, and 100 mM NaCl, pH 8.5 at 22 °C.
Tabatabaei Dakhili et al.
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pH 8.5
pH 7.5
pH 6.5
substrate does not fit into this cleft cavity and is, therefore, dis-
placed from the protein surface (SI Appendix, Fig. S13). Never-
theless, in all cases large structural changes to the protein are
recorded, indicating that both substrate and protein must un-
dergo significant adjustments to match their shape upon binding.
A
B
0.3
0.2
0.1
0
0.3
0.2
0.1
0
R2 = 0.999
Silyl Ether Condensation Catalyzed by TF-Silα. It is well-established
that hydrolytic enzymes such as proteases and esterases can be
driven in the reverse direction (i.e., condensation) by alteration
of the reaction equilibrium (30, 31). To explore the applicability
of this general concept to the silicateins, the condensation of
silanols and alcohols to give the corresponding silyl ethers was
investigated. For this purpose, the enzymes were lyophilized
and the reactions performed in octane. In the first instance,
condensation of 1-octanol (OcOH, 8) with trimethylsilanol
(TMS-OH, 9) to give the corresponding silyl ether (TMS-OOc,
15; Scheme 2) was studied. Analysis of the reaction mixtures at
various time intervals by GC-MS showed that the desired
product was generated (SI Appendix, Figs. S15 and S16) with
reaction conversions of 20% achieved after 72 h. Control ex-
periments using equimolar amounts of heat-denatured enzyme
or when the enzyme was omitted gave only small amounts of
product (3.6% and 1.1%, respectively), via nonspecific catalysis
or the uncatalyzed reaction.
6
7
8
9
10
11
0
100 200 300 400 500
TBDMS-ONp concentration (μM)
pH
Fig. 4. (A) Graph of the rate of hydrolysis of 1 catalyzed by TF-Silα against
pH with an initial 1 concentration of 0.05 mM. (B) Graph of initial reaction
velocity for the hydrolysis of 1 catalyzed by TF-Silα against a range of initial
substrate concentrations. The plotted data are obtained after subtraction of
the rate of background hydrolysis and calibrated using data from SI Ap-
pendix, Figs. S4 and S5. The best fit Michaelis–Menten curve (dotted line)
and associated R2 value are also shown for the pH 8.5 data.
of the enzyme is relatively wide, which may allow it accommo-
date these larger molecules.
To rationalize the experimental observations, molecular dy-
namics (MD) simulations with CHARMM were carried out us-
ing a homology model constructed from the known crystallographic
structure of cathepsin L (28). The silicatein model structure bound
to substrate 1 was created and two sets of MD calculations were
performed for a period of 1 ns, one with constrained substrate ge-
ometry and one fully relaxed structure. After completion of the
calculations, the final snapshot of the latter model showed that the
substrate is bound to the enzyme in close proximity to the active site
catalytic triad (Fig. 6 and SI Appendix, Figs. S10–S14). Here, the
binding cavity appears as a wide cleft that is able to accommodate
large molecules. The model shows that 1 is oriented such that the Si
atom and its substituent alkyl groups point toward the catalytic
residues His165 and Ser26. The 4-nitrophenyl group points outward
to the solvent and fits between the protein backbone and the Arg146
residue, which projects over the substrate (Fig. 6A). The proximity
of the phenyl ring face of 1 with the guanidinium terminal of the Arg
residue (∼3 Å) suggests the intriguing possibility of a cation–π in-
teraction (29), which may contribute toward the binding of a hy-
drophobic substrate to the relatively polar binding site.
To optimize the condensation reaction, a number of param-
eters were investigated. It has previously been reported that
when used in organic solvents, enzymes lyophilized from buffers
at different pH values gave different activities due to the re-
tention of the protonation state related to that pH before ly-
ophilization (32). This effect was investigated for the formation
of 15, using TF-Silα lyophilized from buffered ammonium bi-
carbonate at pH 7, 8, and 9 (SI Appendix, Fig. S18). It was found
that the highest conversion was observed with the enzyme sample
lyophilized from pH 7. The effect of reactant stoichiometry was
then investigated and a 5 mol. eq. excess of silanol 9 was found to
give the best conversion (SI Appendix, Fig. S19). Much lower
conversions were observed with larger excesses of silanol, possibly
due to the apparent substrate inhibition noted above. Using neat
octanol as the solvent resulted in negligible conversions, thought
to be due to the denaturation of the enzyme and displacement of
essential structural water molecules by the alcohol (32).
To maximize the rate of reaction and to test the thermal sta-
bility of the system, the condensations were also carried out at
50 °C and 75 °C. These elevated temperatures gave major im-
provements to conversions and were essentially quantitative after
At the end of the simulation the interatomic distance between
the Si atom and the O atom of Ser26 remains large (∼6 Å) and
suggests further molecular motions would be required to enable
the attack of the catalytic hydroxy group at the Si atom. The MD
simulation where the geometry of 1 is constrained shows that the
TF-Silα
No Enzyme (75°C)
TF-Silα (75°C)
A
B
TF-Silα (Mutant-Ser26Ala)
TF-Silα (Heat Denatured)
TF
No Enzyme (50°C)
TF-Silα (50°C)
No Enzyme
No Enzyme (22°C)
TF-Silα (22°C)
Table 1. Table of Michaelis–Menten constants determined for
Silα and TF-Silα against the model substrates 1–3 (the hydrolysis
of model peptide 7 by cathepsin L is also listed for comparison)
20
18
16
14
12
10
8
100
80
60
40
20
0
Enzyme*
Substrate Km, μM
kcat, min−1 kcat/Km, min−1·μM−1
TF-Silα
TF-Silα
TF-Silα
Silα
1
2
3
1
2
3
7
22.4 2.2 988.3 416.4
8.7 4.5 79.1 19.9
49.8 17.2 92.1 29.4
12.9 2.1 611.1 32.8
55.7 21.2
6
12.3 7.5
2.3 1.4
48.3 6.0
4.6 1.9
4.7 4.0
33.3
4
2
0
Silα
7.5 2.6
76.4 47.2 166.5 89.7
36 1,200
61.5 5.4
0
15
30
45
60
75
0
400
Time (min)
800
1200
Silα
Time (h)
Cathepsin L†
Fig. 5. (A) Graph of the extent of hydrolysis of 2 over time catalyzed by
TF-Silα, its Ser26Ala mutant, heat-denatured TF-Silα, and the TF protein
alone. Assays are conducted under the same conditions as noted for Fig. 3.
(B) Graph of percentage conversion to silyl ether 15 against reaction time at
three different temperatures.
*Assays for silicateins performed in 5% vol/vol 1,4-dioxane, 50 mM Tris, and
100 mM NaCl, pH 8.5.
†Assay performed in 5% vol/vol N,N-dimethylformamide and 100 mM phos-
phate buffer, pH 6.0 (data taken from ref. 25).
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A
B
Arg146
Substrate Product R1
R2
TES TES
n
1
1
1
2
2
2
3
3
3
Product ratio (%)a Conversion (%)a
His165
Ser26
21
22
23
24
25
26
27
28
29
2.0 0.05
23.5 2.9
74.3 1.1
0.8 0.6
0.75 0.02
8.5 1.0
18
18
18
19
19
19
20
20
20
H
TES
H
TES
26.9 0.42
0.4 0.3
Fig. 6. Images of substrate 1 bound to Silα at the end of the MD simulation
without substrate constraints. (A) Image of the overall protein structure
showing the substrate (green) bound at the large active site cavity and the
overhanging Arg146 (orange). (B) Magnification of the area around the
bound substrate, with the substrate (green with the silicon atom in yellow),
Ser26 (magenta), His165 (blue), and Arg146 (orange). Other residues within 5 Å
of the substrate are shown in gray. Asn185 is located behind His165 and is not
visible from this perspective.
TES TES
H
TES
H
18.4 6.8
80.6 23.3
1.1 0.009
15.6 0.03
83.1 1.6
9.4 3.4
TES
41.2 11.9
0.4 0.01
6.0 0.01
32.1 0.6
TES TES
H
TES
H
TES
Scheme 3. Regioselective silyl ether synthesis by silanol condensation. aAfter
24 h using TF-Silα lyophilized with lyoprotectant mixture, 5 mol eq 11 relative
to diol, 75 °C.
72 h at 75 °C (Fig. 5B). In comparison, control experiments
where the enzyme was omitted gave <9% conversion. The ability
of the protein to function at such elevated temperatures is no-
table but conforms with previous reports that nonpolar organic
solvents improve the stability of enzymes through hydrophobic
confinement and thus suppression of denaturation (33, 34).
Another factor that was investigated was the addition of lyo-
protectants such as potassium salts and the crown ether
18-crown-6 (18C6) before enzyme lyophilization (33, 35, 36).
When TF-Silα was colyophilized with KCl and 18C6, then used
for the condensation of 15, conversions could be further improved
compared with when these additives were omitted (SI Appendix,
Figs. S20 and S21). Using these optimized conditions, the con-
densation with octanol 8 with further silanol examples, dime-
thylphenylsilanol (DMPS-OH, 10) and triethylsilanol (TES-OH,
11), were then performed to give the corresponding ethers 16 and
17 (Scheme 2 and SI Appendix, Figs. S22–S25). In all cases >80%
conversions were achieved, demonstrating the use of this enzyme
as a silyl ether condensation catalyst.
responding ethoxysilanes was investigated. Such intermolecular
transetherifications would be a useful route to the silylation of
more valuable substrates using readily available silyl donors
while avoiding the use of chlorosilanes. To demonstrate this
approach, TMS-OEt, DMPS-OEt, and TES-OEt (12–14, Scheme 2)
were used as silyl donors for the silylation of octanol 8 with TF-Silα
catalysis under the optimized conditions previously used for the
condensation reactions (lyoprotectants added, n-octane, 75 °C).
Analysis of the reaction products showed that the silyl groups
could be successfully transferred (SI Appendix, Figs. S26–S31).
However, the reactions were slower compared with the analo-
gous condensation reactions and generally gave poorer conver-
sions (Scheme 2).
Regioselective Silylation. The differentiation of hydroxy groups
remains an important step toward the chemical synthesis of
complex molecules. Such regioselectivity is typically achieved
through the selective deprotection of persilylated substrates (6,
37) but necessitates wasteful global protection beforehand. Di-
rect, selective silylation would circumvent the deprotection step
and would therefore be more efficient. As an initial investiga-
tion into the regioselectivity of biocatalytic silylation, model
4-(ω-hydroxyalkyl)phenol substrates 18–20 that each possess a
phenolic and aliphatic alcohol (Scheme 3) were subjected to
TF-Silα catalyzed silylation with TES-OH (11). For each of these
dihydroxy substrates, analysis of the reaction mixtures indicated
the formation of all three possible products (21–29, Scheme 3
and SI Appendix, Figs. S32–S34). Notably, although the enzyme
eventually catalyzed the silylation of both hydroxy groups, in all
cases there is an initial preference for the phenolic group. For
Silyl Transfer by Transetherification. A major limitation of using
silanols for condensation is their propensity to form disiloxanes.
As an alternative, the transfer of the silyl group from their cor-
Substratea Abbreviationb R1 R2 R3 R4 Product Time (h) Conv.(%)c
9
TMS-OH
Me Me Me
H
H
H
15
16
17
15
16
17
72
48
72
72
48
72
99.0 9.6d
83.0 5.7e
88.6 8.5
77.9 7.5
50.0 9.4e
34.0 1.5
10
11
12
13
14
DMPS-OH Me Me Ph
TES-OH
Et Et Et
TMS-OEt
Me Me Me Et
DMPS-OEt Me Me Ph Et
TES-OEt Et Et Et Et
Substrate R1
R2 Abbreviationa
Scheme 2. Silyl ether synthesis by silanol condensation or ethoxysilane
transetherification. aFive mole equivalents of substrates used relative to
octanol. bDMPS, dimethylphenylsilyl; TES, triethylsilyl; TMS, trimethylsilyl. cAs
determined by GC-MS quantification. Reaction using TF-Silα lyophilized from
the buffered lyoprotectant mixture for 72 h at 75 °C. dReaction using TF-Silα
lyophilized from NH3HCO3 buffer only at 75 °C, shown for comparison. eMaximum
conversion achieved after 48 h.
30
31
O
O-
Piv-ONp
NH NH2
Piv-NHNp
Scheme 4. Enzyme catalyzed hydrolysis of 4-nitrophenyl pivaloate and
pivalamide. aNp, 4-nitrophenyl; Piv, pivaloyl.
Tabatabaei Dakhili et al.
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E5289

7, TF-Silα (pH 8.5)
1, TF-Silα (pH 8.5)
1, TF-Silα (pH 8.5)
A
B
C
1, No Enzyme (pH 8.5)
30, TF-Silα (pH 8.5)
30, No Enzyme (pH 8.5)
31, TF-Silα (pH 8.5)
31, No Enzyme (pH 8.5)
7, Chymotrypsin (pH 8.5)
7, Cathepsin L (pH 6.8)
7, No Enzyme (pH 8.5)
7, No Enzyme (pH 6.8)
1, Chymotrypsin (pH 8.5)
1, Cathepsin L (pH 6.8)
1, No Enzyme (pH 6.8)
1, No Enzyme (pH 8.5)
35
15
10
5
35
30
25
20
15
10
5
30
25
20
15
10
5
0
0
0
0
40
80
120 160
0
50
100
150
0
50
100
150
Time (Min)
Time (min)
Time (min)
Fig. 7. Graphs of the concentration of chromophores (4-nitroaniline and 4-nitrophenoxylate ion) generated against time for the hydrolysis of: (A) TBDMS-
ONp (1), Piv-ONp (30), or Piv-NHNp (31) catalyzed by TF-Silα; (B) Cbz-Phe-Arg-NHNp (7) catalyzed by TF-Silα, chymotrypsin, or cathepsin L; and (C) 1 catalyzed
by TF-Silα, chymotrypsin, or cathepsin L.
example, the silylation of 19 gave a 52% substrate conversion
after 24 h, of which over 80% was the aryl siloxane 26. The al-
iphatic siloxane 25 comprised most of the remainder of the
product, with only trace amounts (∼1%) of the disilylated ma-
terial 24. In contrast, negative control experiments where the
enzyme was omitted gave very low total conversions and silyla-
tion of the aliphatic alcohol was preferred (∼0.5% of 25 was
produced after 24 h), as would be expected due to the higher
nucleophilicity of the aliphatic alcohol under these conditions.
This general trend was repeated for the other substrates that
were tested.
In contrast, chymotrypsin and cathepsin L only catalyzed the
hydrolysis of siloxane 1 at low levels, which is attributable to
nonspecific catalysis (Fig. 7C and Table 2). In all cases, no hy-
drolysis of the amide 31 was detectable, although chymotrypsin
demonstrated a good level of hydrolytic activity against the ester
30 (Table 2), in agreement with the known promiscuous esterase
activity of this enzyme even against bulky substrates (39).
Conclusions
In summary, the production of two recombinant silicateins and a
systematic survey into their reactivity against various organo-
siloxanes has been conducted. In the process, a high-throughput
colorimetric assay for silyl ether hydrolysis was developed. This
assay showed that the enzymes are able to catalyze the hydrolysis
of a range of silyl ethers, including those with very bulky sub-
stituents, albeit at a relatively slow rate. The silicateins display
good activity from pH 7.5–10.0, with substrate inhibition ob-
served below this pH range. Supporting MD modeling of the
enzyme with a model substrate showed that substrate fits into the
binding cavity, with the silicon center oriented toward the pre-
sumed catalytic residues. The binding of these large substrates is
possible as a result of large conformational changes in the pro-
tein and substrate distortion. These results are consistent with
the proposed hydrolytic mechanism that is used by enzymes
possessing a catalytic triad motif.
The silicateins were also shown to catalyze the condensation of
organosilanols and alcohols to give the corresponding silyl ethers
when used in organic solvents. Furthermore, they can catalyze
transetherifications, where the silyl group from one silyl ether
may be transferred to a recipient alcohol. Notably, when pre-
sented with a substrate bearing both aliphatic and aromatic hy-
droxy groups the enzyme preferentially catalyzes the silylation of
the latter group, in clear contrast to nonenzymatic silylations.
Comparisons with Small-Molecule Catalysts. Using the condensation
reaction of octanol 8 and TMS-OH (9) to form 15 as a model
reaction, the effectiveness of common catalysts such as imidazole,
triethylamine, and histidine were compared. At similar catalyst
loadings, these bases gave negligible conversions (<0.5% after
24 h) compared with the enzyme-catalyzed reaction (17%, SI
Appendix, Fig. S35). These small-molecule catalysts also gave low
regioselectivity with the dihydroxy compound 19. Taking imidaz-
ole as an example, if a large amount of catalyst was used (3 mol eq
relative to alcohol) and the reaction was halted at an early time
point (after 5 h), the aliphatic silyl ether 24 and disilylated 25 were
clearly the dominant products (SI Appendix, Fig. S36).
These results are significant because they show that the en-
zymatic catalysis is not simply due to the presence of basic func-
tional groups, and that the enzymatic reaction results in contrasting
regioselectivity compared with small-molecule catalysis.
Esterase and Protease Activity. Since Silα has a high degree of
homology with the protease cathepsin L and employs a catalytic
triad common to many other hydrolytic enzymes, its esterase and
protease activity was surveyed. Here, analogs of 1 where the
dimethylsiloxy moiety was replaced with an ester or amide (30 and
31 respectively, Scheme 4) were tested. Time-course experiments
for the hydrolysis of these substrates catalyzed by TF-Silα and Silα
showed no specific hydrolysis, comparable with control experi-
ments where the enzyme was omitted (Fig. 7A and Table 2). Both
the candidate enzymes therefore seem to have negligible esterase
or amidase activity against such analogous substrates.
Table 2. Table of percentage conversions for the enzyme
catalyzed hydrolysis of the various substrates
Net % conversion
Enzyme
1
30
31
7
When tested against dipeptide 7 both silicateins also displayed
negligible activity in comparison with both chymotrypsin and
cathepsin L, which readily hydrolyzed this dipeptide (Fig. 7B and
Table 2). These results are consistent with a previous report where
a Cys→Ser mutation in the cathepsin L active site largely abolishes
protease activity (38). However, it does not exclude the possibility
that Silα may have protease activity against very specific sequences
such as in the autolysis of its own propeptide during maturation of
the enzyme. Indeed, previous work with the silicatein proenzyme
has shown that self-cleavage is possible (15).
TF-Silα
Silα
Chymotrypsin
Cathepsin L*
19.2 1.2
21.4 0.3
6.9 0.05
2.5 1.3
<0.01 0.0 <0.01 0.0
1.3 0.8 <0.01 0.0
17.5 7.1 <0.01 0.0
<0.01 0.0
<0.01 0.0
21.4 0.5
26.7 1.8
1.6 1.7
<0.2 0.01
Net conversions are calculated after subtraction of background hydrolysis.
All reactions were performed for 4 h with 0.00067 mol eq enzyme with
0.2 mM substrate in 5% vol/vol dioxane, 50 mM Tris, and 100 mM NaCl.
*Assay was conducted at pH 6.8, the optimum for this enzyme; all other
assays were performed at pH 8.5.
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Tabatabaei Dakhili et al.

Despite sequence similarities with the cathepsins, the silica-
teins seem to exhibit no significant protease or esterase activity
when tested against analogous substrates. The silicateins thus
represent an example of divergent evolution where an existing
ancestral enzyme has evolved to catalyze reactions in a new
“niche” of chemical space.
Further work will be needed to develop an in-depth under-
standing of the structure and mechanism of this family of en-
zymes. Such structural elucidation of the silicateins remains a
significant challenge (18) due to the low protein production yield
and their hydrophobicity, but it is anticipated that the work
reported here will provide a solid foundation toward this end and
to the wider goal of using enzymes for applied biocatalysis.
The results reported herein therefore suggest that the silica-
teins are promising candidates for future development into ef-
ficient and selective biocatalysts for organosiloxane chemistry.
By providing a new chemical context (e.g., the condensation of
organic silyl ethers in organic solvents), they may be considered
prototype “silyl etherase” enzymes that can be subjected to fur-
ther evolutionary optimization (40). Indeed, powerful directed
evolution strategies are now available to generate highly specific
and robust biocatalysts for applications in the production of fine
chemicals and functional materials (10, 41). It is envisaged that
such biocatalysts could be used in the chemical synthesis of
complex molecules, where they could be used to selectively in-
troduce or cleave silyl protecting groups (6) or to recycle these
relatively expensive silyl groups by transetherification (13). In the
area of materials chemistry, they could be applied for the syn-
thesis of silicone polymers from nonhalogenated feedstocks (42).
Materials and Methods
The proteins were heterologously produced from synthetic genes in E. coli
and isolated using standard procedures. The silyl ether substrates were
synthesized from the corresponding chlorosilanes, silyl triflates, or silazanes.
The GC-MS analyses of the reactions were performed by comparison and
calibration with chemically synthesized samples. Full details for the materials
and methods are given in SI Appendix. The tabulated CD data are given in
Dataset S1.
ACKNOWLEDGMENTS. We thank Emily I. Sparkes for technical assistance
and Prof. Peter G. Taylor (Open University, Milton Keynes, United Kingdom)
for assistance with the chemical data analysis. This work was supported by
Engineering and Physical Sciences Research Council Grants EP/K011685/1 and
EP/K031465/1 (to L.S.W.), Biotechnology and Biological Sciences Research
Council Doctoral Training Partnership Graduate Studentship BB/J014478/1
(to S.A.C.), and a graduate studentship from the Tertiary Education Trust
Fund of Nigeria (to A.S.F.).
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Published online June 19, 2017
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