Recombinant silicateins as model biocatalysts inorganosiloxane chemistryS. 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 (phylumPorifera) is unique in nature for catalyzing the formation ofinorganic silica structures, which the organisms incorporate intotheir 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 thisimportant class of compounds, their catalysis of Si–O bond hydro-lysis and condensation was investigated with a range of modelorganosilanols and silyl ethers. The enzymes’ kinetic parameterswere obtained by a high-throughput colorimetric assay based onthe hydrolysis of 4-nitrophenyl silyl ethers. These assays showedunambiguous catalysis with kcat/Km values on the order of2–50 min−1 μM−1. Condensation reactions were also demonstratedby the generation of silyl ethers from their corresponding silanolsand alcohols. Notably, when presented with a substrate bearingboth aliphatic and aromatic hydroxy groups the enzyme preferen-tially silylates the latter group, in clear contrast to nonenzymaticsilylations. Furthermore, the silicateins are able to catalyze trans-etherifications, where the silyl group from one silyl ether may betransferred to a recipient alcohol. Despite close sequence homol-ogy to the protease cathepsin L, the silicateins seem to exhibit nosignificant protease or esterase activity when tested against anal-ogous substrates. Overall, these results suggest the silicateins arepromising candidates for future elaboration into efficient and se-lective biocatalysts for organosiloxane chemistry.

silicatein | biocatalysis | organosilicon | organosiloxane | silyl ether

The organosiloxanes, compounds containing C–Si–O moieties,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 andconsumer products for a variety of purposes such as bulkingagents, separation media, protective coatings, lubricants, emul-sifiers, and adhesives (1–3). Their use as auxiliaries in thechemical synthesis of complex molecules is also long-established(4–6). However, the production and synthetic manipulation ofthese compounds are almost entirely dependent on chlorosilanefeedstocks, which are environmentally undesirable and energy-intensive to produce (7, 8). Furthermore, organosiloxanes, whichare entirely anthropogenic in origin, are now known to be per-sistent environmental contaminants because little attempt ismade to recover and recycle them (3). Synthetic routes that use,and ultimately recycle, siloxanes and silanols as alternativeswould in principle be more environmentally sound.One possible strategy toward improved sustainability is to

harness enzymes for chemical processing. Such biocatalysts areattractive because they offer highly efficient synthesis in terms ofyields and regio- and stereospecificity, together with an ability topromote reactions under mild conditions and a minimal relianceon halogenated or metallic feedstocks (9, 10). The use of en-zymes to manipulate the Si–O bond would therefore potentiallyoffer more sustainable routes to the synthesis of many com-pounds, as well as for the eventual recycling and reuse of orga-

nosiloxanes. Several attempts have been made to use hydrolyticenzymes such as lipases and proteases for the hydrolysis andcondensation of the Si–O bond (11–13). Although they clearlydemonstrate the feasibility of the general concept, the range ofenzymes tested have so far met with only limited success inregard 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 thesilicateins to catalyze the polymerization of soluble silicates intosilica (14–16). The primary sequences of these enzymes havebeen reported and they bear a remarkable homology with pro-teases of the cathepsin family, with ∼65% sequence similaritiesand ∼50% sequence identities relative to cathepsin L. Both en-zymes share a similar Xaa–His–Asn catalytic triad at their activesite, although in the silicateins a Ser residue occupies the Xaaposition rather than Cys in cathepsin L. Previous reports haveshown that silicatein-α (Silα), the prototypical member of thisfamily, 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 pointfor further elaboration into practical biocatalysts in organo-siloxane chemistry. This paper outlines the performance of het-erologously produced Silα for both the hydrolysis and condensationof a range of model organosiloxanes. In the process, the devel-opment of a colorimetric high-throughput screening methodfor silyl ether bond hydrolysis based on the 4-nitrophenoxylate

Significance

Organosiloxanes are components in a huge variety of con-sumer products and play a major role in the synthesis of finechemicals. However, their synthetic manipulation primarilyrelies on the use of chlorosilanes, which are energy-intensive toproduce and environmentally undesirable. Synthetic routesthat operate under ambient conditions and circumvent theneed for chlorinated feedstocks would therefore offer a moresustainable route for producing this class of compounds. Here,a systematic survey is reported for the silicatein enzyme, whichis able to catalyze the hydrolysis, condensation, and exchangeof the silicon–oxygen bond in a variety of organosiloxanesunder environmentally benign conditions. These results sug-gest that silicatein is a promising candidate for development ofselective and efficient biocatalysts for organosiloxane chemistry.

Author contributions: S.Y.T.D., S.P.d.V., and L.S.W. designed research; S.Y.T.D., S.A.C., andA.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 | Published online June 19, 2017 | E5285–E5291

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chromophore is also reported. Additionally, the protease and es-terase activity of Silα against analogous substrates is described.

Results and DiscussionProduction and Characterization of Silα. To acquire this enzyme, asynthetic vector containing cDNA encoding for the mature wild-type Silα from Suberites domencula, fused to an N-terminalhexahistidine tag and codon optimized for expression in Escherichiacoli, was used. It is known that the mature form of the protein ishighly 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 enhancesolubility and folding. Sequences encoding for GST, thioredoxin,small ubiquitin-like modifier, maltose binding protein, or triggerfactor (TF) were inserted between the hexahistidine tag and Silαand the genes transformed into a variety of E. coli BL21(DE3)strains including Arctic-Express and Origami. Expression trialswere 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-Silαfusion 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 recoveryof sufficient levels of the soluble protein for some further studies.Both the TF-Silα fusion and the solubilized wild-type Silα werethen 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 specialprecautions. Size-exclusion chromatography of both these pro-teins in isolated form showed a single well-defined species (SIAppendix, Fig. S1A). The CD spectrum of each protein showedclear secondary structural features, indicating the proteins werenot denatured or disordered (Fig. 2 and SI Appendix, Fig. S2).The spectra of the mutant and unmodified protein essentiallyoverlap, showing that the mutation did not affect its overallfolding. The relative proportions of secondary structures werealso calculated from these spectra and were found to be within4% of the values calculated from combining the crystallograph-ically derived data of a cathepsin-silicatein chimera (18) and TF(SI Appendix, Tables S1–S3).

In contrast, Silα could be maintained in a homogeneous stateonly at dilute concentrations (micromolar range), which wereinsufficient for CD measurements. However, analysis by non-denaturing gel electrophoresis for Silα clearly showed a singleband, indicating nonaggregation (SI Appendix, Fig. S1B). Theprotein advanced through the gel at a similar rate as the 25 kDareference protein, suggesting that it was of approximately similarsize, globular, and monomeric.

Determination of Enzymatic Activity. To confirm that the twocandidate proteins (Silα and TF-Silα) were catalytically compe-tent, their hydrolytic activity against TEOS was tested and theamount of precipitated silica quantified by the previouslyreported silicomolybdic acid assay (17). The amount of silicaproduced upon exposure of silicic acid (derived from acid hy-drolysis of TEOS) to these enzymes was also measured. Bothassays demonstrated that the enzymes were active for both thehydrolysis 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 dueto 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) andtests with trypsin and papain that are known to reject TEOS as asubstrate (11, 17).

High-Throughput Colorimetric Assays for Silyl Ether Hydrolysis. Toinvestigate the scope of these recombinant silicateins in themanipulation of a wider range of siloxanes, it was necessary todevelop a new screening methodology for this class of com-pounds, because the silicomolybdic acid assay is incompatiblewith organosilanes (compounds with C–Si bonds). For this pur-pose, a series of 4-nitrophenoxy silyl ethers 1–3 was synthesizedfor use as model substrates (Scheme 1). Here, it was envisagedthat hydrolysis of the Si–O bond would result in the releaseof the corresponding silanols 4–6 and the strongly absorbingnitrophenoxylate ion, which can be quantified by UV-visible(UV-Vis) spectrophotometry.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-MSanalysis. It was further observed that the assay in fact generated ayellow 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 anycatalyst an appreciable rate of product formation is detectable.Nevertheless, assays that used the fully competent enzyme alwaysgave enhanced rates of hydrolysis compared with the controlexperiments or the uncatalyzed reaction.

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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 isolatedproteins. Also included above each gel image are the schematic represen-tations of the protein constructs.

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Fig. 2. CD spectra plots of molar ellipticity against wavelength for TF-Silαand TF-Silα(Ser26Ala).

E5286 | www.pnas.org/cgi/doi/10.1073/pnas.1613320114 Tabatabaei Dakhili et al.

Taken together, these results demonstrate the feasibility ofusing 4-nitrophenoxy silyl ethers as a high-throughput assay andshow that both enzymes accelerated silyl ether bond hydrolysissignificantly above that of background uncatalyzed hydrolysis.

pH Dependence of TF-Silα Hydrolytic Activity. To investigate theeffect of pH on enzymatic silyl ether hydrolysis the initial rates ofhydrolysis of 1 catalyzed by TF-Silα over pH 6.5–10.5 were de-termined. Here, the initial rates of reaction were used as ameasure 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 pHrange with a maximum at pH ∼10 (Fig. 4A), consistent to theoptimum pH range of “alkaline” serine proteases (20–22). Thisoptimum 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 theacyl-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 ofmechanisms (23, 24). Of the proposed mechanisms, the classicalsubstrate 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, andthere are no known compounds bearing C–Si bonds of biologicalorigin (3). Models where the increasing amounts of substrateresult in the formation of noncatalytic conformations of the

enzyme could be invoked, although how this effect is related tothe pH is unclear at present.

Kinetic Analysis of TF-Silα and Silα-Catalyzed Silyl Ether Hydrolysis.To further characterize this enzyme-catalyzed hydrolysis, a seriesof assays were conducted to extract the Michaelis–Menten kcatand Km values (Table 1 and SI Appendix, Figs. S7–S9). Based onthe pH study above, these assays were performed at pH 8.5 as anacceptable compromise between relatively good enzyme activity,low levels of background (uncatalyzed) hydrolysis, and avoidanceof substrate inhibition.In general, the binding of both candidate proteins to all of the

substrates was relatively weak, with Km values in the micromolarrange. However, it did not follow in the expected trend of de-creasing Km with increasing substrate steric bulk. The TDS silylether 2 gave a lower Km than the smaller tert-butyldimethylsilyl(TBDMS) analog across both TF-Silα and Silα. This observationsuggests that the enzymes display some selectivity in regard tosubstrate shape that is not simply a function of overall steric bulkof the substrate. The kcat values also followed this general trend,with substrate 2 giving the lowest turnover.The overall catalytic efficiency (kcat/Km) did follow a trend of

falling with increasing bulk of the substrates. These values werecomparable with the data from cathepsin L against its standardCbz-Phe-Arg-NHNp dipeptide nitrophenylanilide substrate 7(25), suggesting the kinetics data obtained for the silicateinswere plausible.

Ser26Ala Mutation at the TF-Silα Active Site. To confirm that thecatalysis did indeed involve the serine residue at the active site,the Ser26Ala mutant of TF-Silα was then tested. Using TEOS asa 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 agreementwith a previous report where the equivalent mutation in wild-type Silα from Tethya aurantia resulted in the abolition of specificcatalysis (26). When tested against the chromogenic substrate 2 asimilar result was obtained, with the unmodified TF-Silα result-ing in a clear positive result whereas only low activity was foundin the mutant, comparable to the other control experiments (Fig.5A). Although greatly reduced in all cases, total loss of catalysisis never observed due to residual nonspecific catalysis, aspreviously noted.

Molecular Dynamics Modeling of Silα Binding with TBDMS-ONp. Itwas notable that the enzymes were able to process large non-polar organosiloxanes, albeit at a low rate. There is currently noexperimentally derived structure for any of the silicateins, butprevious models (27) have suggested that the active site pocket

Substrate Abbreviationa R1 R2 R3 Product

1 TBDMS-ONp Me Me tBu 4

2 TDS-ONp Me Me Thxa 53 TIPS-ONp iPr iPr iPr 6

Scheme 1. Hydrolysis of model 4-nitrophenoxy silyl ethers. aNp, 4-nitrophenyl;TBDMS, tert-butyldimethylsilyl; TDS, thexyldimethylsilyl; Thx, thexyl TIPS,tri(iso-propyl)silyl.

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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 at405 nm corresponding to the 4-nitrophenoxylate anion (calibrated using data from SI Appendix, Fig. S4). Assays are conducted with 0.00067 mol eq of enzymerelative 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. PNAS | Published online June 19, 2017 | E5287

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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 crystallographicstructure of cathepsin L (28). The silicatein model structure boundto substrate 1 was created and two sets of MD calculations wereperformed for a period of 1 ns, one with constrained substrate ge-ometry and one fully relaxed structure. After completion of thecalculations, the final snapshot of the latter model showed that thesubstrate is bound to the enzyme in close proximity to the active sitecatalytic triad (Fig. 6 and SI Appendix, Figs. S10–S14). Here, thebinding cavity appears as a wide cleft that is able to accommodatelarge molecules. The model shows that 1 is oriented such that the Siatom and its substituent alkyl groups point toward the catalyticresidues His165 and Ser26. The 4-nitrophenyl group points outwardto the solvent and fits between the protein backbone and the Arg146residue, which projects over the substrate (Fig. 6A). The proximityof the phenyl ring face of 1 with the guanidinium terminal of the Argresidue (∼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.At the end of the simulation the interatomic distance between

the Si atom and the O atom of Ser26 remains large (∼6 Å) andsuggests further molecular motions would be required to enablethe attack of the catalytic hydroxy group at the Si atom. The MDsimulation where the geometry of 1 is constrained shows that the

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 arerecorded, indicating that both substrate and protein must un-dergo significant adjustments to match their shape upon binding.

Silyl Ether Condensation Catalyzed by TF-Silα. It is well-establishedthat hydrolytic enzymes such as proteases and esterases can bedriven in the reverse direction (i.e., condensation) by alterationof the reaction equilibrium (30, 31). To explore the applicabilityof this general concept to the silicateins, the condensation ofsilanols and alcohols to give the corresponding silyl ethers wasinvestigated. For this purpose, the enzymes were lyophilizedand 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 atvarious time intervals by GC-MS showed that the desiredproduct was generated (SI Appendix, Figs. S15 and S16) withreaction conversions of 20% achieved after 72 h. Control ex-periments using equimolar amounts of heat-denatured enzymeor when the enzyme was omitted gave only small amounts ofproduct (3.6% and 1.1%, respectively), via nonspecific catalysisor the uncatalyzed reaction.To optimize the condensation reaction, a number of param-

eters were investigated. It has previously been reported thatwhen used in organic solvents, enzymes lyophilized from buffersat 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 formationof 15, using TF-Silα lyophilized from buffered ammonium bi-carbonate at pH 7, 8, and 9 (SI Appendix, Fig. S18). It was foundthat the highest conversion was observed with the enzyme samplelyophilized from pH 7. The effect of reactant stoichiometry wasthen investigated and a 5 mol. eq. excess of silanol 9 was found togive the best conversion (SI Appendix, Fig. S19). Much lowerconversions were observed with larger excesses of silanol, possiblydue to the apparent substrate inhibition noted above. Using neatoctanol as the solvent resulted in negligible conversions, thoughtto be due to the denaturation of the enzyme and displacement ofessential 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 at50 °C and 75 °C. These elevated temperatures gave major im-provements to conversions and were essentially quantitative after

Table 1. Table of Michaelis–Menten constants determined forSilα and TF-Silα against the model substrates 1–3 (the hydrolysisof model peptide 7 by cathepsin L is also listed for comparison)

Enzyme* Substrate Km, μM kcat, min−1 kcat/Km, min−1·μM−1

TF-Silα 1 22.4 ± 2.2 988.3 ± 416.4 55.7 ± 21.2TF-Silα 2 8.7 ± 4.5 79.1 ± 19.9 12.3 ± 7.5TF-Silα 3 49.8 ± 17.2 92.1 ± 29.4 2.3 ± 1.4Silα 1 12.9 ± 2.1 611.1 ± 32.8 48.3 ± 6.0Silα 2 7.5 ± 2.6 61.5 ± 5.4 4.6 ± 1.9Silα 3 76.4 ± 47.2 166.5 ± 89.7 4.7 ± 4.0Cathepsin L† 7 36 1,200 33.3

*Assays for silicateins performed in 5% vol/vol 1,4-dioxane, 50 mM Tris, and100 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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Fig. 4. (A) Graph of the rate of hydrolysis of 1 catalyzed by TF-Silα againstpH with an initial 1 concentration of 0.05 mM. (B) Graph of initial reactionvelocity for the hydrolysis of 1 catalyzed by TF-Silα against a range of initialsubstrate concentrations. The plotted data are obtained after subtraction ofthe 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.

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Fig. 5. (A) Graph of the extent of hydrolysis of 2 over time catalyzed byTF-Silα, its Ser26Ala mutant, heat-denatured TF-Silα, and the TF proteinalone. Assays are conducted under the same conditions as noted for Fig. 3.(B) Graph of percentage conversion to silyl ether 15 against reaction time atthree different temperatures.

E5288 | www.pnas.org/cgi/doi/10.1073/pnas.1613320114 Tabatabaei Dakhili et al.

72 h at 75 °C (Fig. 5B). In comparison, control experimentswhere the enzyme was omitted gave <9% conversion. The abilityof the protein to function at such elevated temperatures is no-table but conforms with previous reports that nonpolar organicsolvents improve the stability of enzymes through hydrophobicconfinement 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 ether18-crown-6 (18C6) before enzyme lyophilization (33, 35, 36).When TF-Silα was colyophilized with KCl and 18C6, then usedfor the condensation of 15, conversions could be further improvedcompared 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 and17 (Scheme 2 and SI Appendix, Figs. S22–S25). In all cases >80%conversions were achieved, demonstrating the use of this enzymeas a silyl ether condensation catalyst.

Silyl Transfer by Transetherification. A major limitation of usingsilanols for condensation is their propensity to form disiloxanes.As an alternative, the transfer of the silyl group from their cor-

responding ethoxysilanes was investigated. Such intermoleculartransetherifications would be a useful route to the silylation ofmore valuable substrates using readily available silyl donorswhile avoiding the use of chlorosilanes. To demonstrate thisapproach, TMS-OEt, DMPS-OEt, and TES-OEt (12–14, Scheme 2)were used as silyl donors for the silylation of octanol 8 with TF-Silαcatalysis under the optimized conditions previously used for thecondensation reactions (lyoprotectants added, n-octane, 75 °C).Analysis of the reaction products showed that the silyl groupscould 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 groupsremains an important step toward the chemical synthesis ofcomplex molecules. Such regioselectivity is typically achievedthrough the selective deprotection of persilylated substrates (6,37) but necessitates wasteful global protection beforehand. Di-rect, selective silylation would circumvent the deprotection stepand would therefore be more efficient. As an initial investiga-tion into the regioselectivity of biocatalytic silylation, model4-(ω-hydroxyalkyl)phenol substrates 18–20 that each possess aphenolic and aliphatic alcohol (Scheme 3) were subjected toTF-Silα catalyzed silylation with TES-OH (11). For each of thesedihydroxy substrates, analysis of the reaction mixtures indicatedthe formation of all three possible products (21–29, Scheme 3and SI Appendix, Figs. S32–S34). Notably, although the enzymeeventually catalyzed the silylation of both hydroxy groups, in allcases there is an initial preference for the phenolic group. For

Substrate Product R1 R2 n Product ratio (%)a Conversion (%)a

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Scheme 3. Regioselective silyl ether synthesis by silanol condensation. aAfter24 h using TF-Silα lyophilized with lyoprotectant mixture, 5 mol eq 11 relativeto diol, 75 °C.

Substratea Abbreviationb R1 R2 R3 R4 Product Time (h) Conv.(%)c

9 TMS-OH Me Me Me H 15 72 99.0 ± 9.6d

10 DMPS-OH Me Me Ph H 16 48 83.0 ± 5.7e

11 TES-OH Et Et Et H 17 72 88.6 ± 8.5

12 TMS-OEt Me Me Me Et 15 72 77.9 ± 7.5

13 DMPS-OEt Me Me Ph Et 16 48 50.0 ± 9.4e

14 TES-OEt Et Et Et Et 17 72 34.0 ± 1.5

Scheme 2. Silyl ether synthesis by silanol condensation or ethoxysilanetransetherification. aFive mole equivalents of substrates used relative tooctanol. bDMPS, dimethylphenylsilyl; TES, triethylsilyl; TMS, trimethylsilyl. cAsdetermined by GC-MS quantification. Reaction using TF-Silα lyophilized fromthe buffered lyoprotectant mixture for 72 h at 75 °C. dReaction using TF-Silαlyophilized fromNH3HCO3 buffer only at 75 °C, shown for comparison. eMaximumconversion achieved after 48 h.

Arg146

Ser26

His165

A B

Fig. 6. Images of substrate 1 bound to Silα at the end of the MD simulationwithout substrate constraints. (A) Image of the overall protein structureshowing the substrate (green) bound at the large active site cavity and theoverhanging Arg146 (orange). (B) Magnification of the area around thebound substrate, with the substrate (green with the silicon atom in yellow),Ser26 (magenta), His165 (blue), and Arg146 (orange). Other residues within 5 Åof the substrate are shown in gray. Asn185 is located behind His165 and is notvisible from this perspective.

Substrate R1 R2 Abbreviationa

30 O O- Piv-ONp

31 NH NH2 Piv-NHNp

Scheme 4. Enzyme catalyzed hydrolysis of 4-nitrophenyl pivaloate andpivalamide. aNp, 4-nitrophenyl; Piv, pivaloyl.

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example, the silylation of 19 gave a 52% substrate conversionafter 24 h, of which over 80% was the aryl siloxane 26. The al-iphatic siloxane 25 comprised most of the remainder of theproduct, with only trace amounts (∼1%) of the disilylated ma-terial 24. In contrast, negative control experiments where theenzyme was omitted gave very low total conversions and silyla-tion of the aliphatic alcohol was preferred (∼0.5% of 25 wasproduced after 24 h), as would be expected due to the highernucleophilicity of the aliphatic alcohol under these conditions.This general trend was repeated for the other substrates thatwere tested.

Comparisons with Small-Molecule Catalysts.Using the condensationreaction of octanol 8 and TMS-OH (9) to form 15 as a modelreaction, the effectiveness of common catalysts such as imidazole,triethylamine, and histidine were compared. At similar catalystloadings, these bases gave negligible conversions (<0.5% after24 h) compared with the enzyme-catalyzed reaction (17%, SIAppendix, Fig. S35). These small-molecule catalysts also gave lowregioselectivity with the dihydroxy compound 19. Taking imidaz-ole as an example, if a large amount of catalyst was used (3 mol eqrelative to alcohol) and the reaction was halted at an early timepoint (after 5 h), the aliphatic silyl ether 24 and disilylated 25 wereclearly 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 contrastingregioselectivity compared with small-molecule catalysis.

Esterase and Protease Activity. Since Silα has a high degree ofhomology with the protease cathepsin L and employs a catalytictriad common to many other hydrolytic enzymes, its esterase andprotease activity was surveyed. Here, analogs of 1 where thedimethylsiloxy moiety was replaced with an ester or amide (30 and31 respectively, Scheme 4) were tested. Time-course experimentsfor the hydrolysis of these substrates catalyzed by TF-Silα and Silαshowed no specific hydrolysis, comparable with control experi-ments where the enzyme was omitted (Fig. 7A and Table 2). Boththe candidate enzymes therefore seem to have negligible esteraseor amidase activity against such analogous substrates.When tested against dipeptide 7 both silicateins also displayed

negligible activity in comparison with both chymotrypsin andcathepsin L, which readily hydrolyzed this dipeptide (Fig. 7B andTable 2). These results are consistent with a previous report wherea Cys→Ser mutation in the cathepsin L active site largely abolishesprotease activity (38). However, it does not exclude the possibilitythat Silαmay have protease activity against very specific sequencessuch as in the autolysis of its own propeptide during maturation ofthe enzyme. Indeed, previous work with the silicatein proenzymehas shown that self-cleavage is possible (15).

In contrast, chymotrypsin and cathepsin L only catalyzed thehydrolysis of siloxane 1 at low levels, which is attributable tononspecific catalysis (Fig. 7C and Table 2). In all cases, no hy-drolysis of the amide 31 was detectable, although chymotrypsindemonstrated a good level of hydrolytic activity against the ester30 (Table 2), in agreement with the known promiscuous esteraseactivity of this enzyme even against bulky substrates (39).

ConclusionsIn summary, the production of two recombinant silicateins and asystematic survey into their reactivity against various organo-siloxanes has been conducted. In the process, a high-throughputcolorimetric assay for silyl ether hydrolysis was developed. Thisassay showed that the enzymes are able to catalyze the hydrolysisof a range of silyl ethers, including those with very bulky sub-stituents, albeit at a relatively slow rate. The silicateins displaygood activity from pH 7.5–10.0, with substrate inhibition ob-served below this pH range. Supporting MD modeling of theenzyme with a model substrate showed that substrate fits into thebinding cavity, with the silicon center oriented toward the pre-sumed catalytic residues. The binding of these large substrates ispossible as a result of large conformational changes in the pro-tein and substrate distortion. These results are consistent withthe proposed hydrolytic mechanism that is used by enzymespossessing a catalytic triad motif.The silicateins were also shown to catalyze the condensation of

organosilanols and alcohols to give the corresponding silyl etherswhen used in organic solvents. Furthermore, they can catalyzetransetherifications, where the silyl group from one silyl ethermay 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 ofthe latter group, in clear contrast to nonenzymatic silylations.

0

5

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15

20

25

30

35

0 40 80 120 160

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n (μM)

Time (min)

1, TF-Silα (pH 8.5)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)

A

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7, TF-Silα (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)

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cent

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M)

Time (Min)

B

0

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15

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1, TF-Silα (pH 8.5)1, Chymotrypsin (pH 8.5)1, Cathepsin L (pH 6.8)1, No Enzyme (pH 6.8)1, No Enzyme (pH 8.5)

C

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 catalyzedby TF-Silα, chymotrypsin, or cathepsin L.

Table 2. Table of percentage conversions for the enzymecatalyzed hydrolysis of the various substrates

Enzyme

Net % conversion

1 30 31 7

TF-Silα 19.2 ± 1.2 <0.01 ± 0.0 <0.01 ± 0.0 <0.01 ± 0.0Silα 21.4 ± 0.3 1.3 ± 0.8 <0.01 ± 0.0 <0.01 ± 0.0Chymotrypsin 6.9 ± 0.05 17.5 ± 7.1 <0.01 ± 0.0 21.4 ± 0.5Cathepsin L* 2.5 ± 1.3 1.6 ± 1.7 <0.2 ± 0.01 26.7 ± 1.8

Net conversions are calculated after subtraction of background hydrolysis.All reactions were performed for 4 h with 0.00067 mol eq enzyme with0.2 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 otherassays were performed at pH 8.5.

E5290 | www.pnas.org/cgi/doi/10.1073/pnas.1613320114 Tabatabaei Dakhili et al.

Despite sequence similarities with the cathepsins, the silica-teins seem to exhibit no significant protease or esterase activitywhen tested against analogous substrates. The silicateins thusrepresent an example of divergent evolution where an existingancestral enzyme has evolved to catalyze reactions in a new“niche” of chemical space.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 oforganic silyl ethers in organic solvents), they may be consideredprototype “silyl etherase” enzymes that can be subjected to fur-ther evolutionary optimization (40). Indeed, powerful directedevolution strategies are now available to generate highly specificand robust biocatalysts for applications in the production of finechemicals and functional materials (10, 41). It is envisaged thatsuch biocatalysts could be used in the chemical synthesis ofcomplex molecules, where they could be used to selectively in-troduce or cleave silyl protecting groups (6) or to recycle theserelatively expensive silyl groups by transetherification (13). In thearea of materials chemistry, they could be applied for the syn-thesis of silicone polymers from nonhalogenated feedstocks (42).

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 asignificant challenge (18) due to the low protein production yieldand their hydrophobicity, but it is anticipated that the workreported here will provide a solid foundation toward this end andto the wider goal of using enzymes for applied biocatalysis.

Materials and MethodsThe proteins were heterologously produced from synthetic genes in E. coliand isolated using standard procedures. The silyl ether substrates weresynthesized from the corresponding chlorosilanes, silyl triflates, or silazanes.The GC-MS analyses of the reactions were performed by comparison andcalibration with chemically synthesized samples. Full details for the materialsand methods are given in SI Appendix. The tabulated CD data are given inDataset S1.

ACKNOWLEDGMENTS. We thank Emily I. Sparkes for technical assistanceand Prof. Peter G. Taylor (Open University, Milton Keynes, United Kingdom)for assistance with the chemical data analysis. This work was supported byEngineering and Physical Sciences Research Council Grants EP/K011685/1 andEP/K031465/1 (to L.S.W.), Biotechnology and Biological Sciences ResearchCouncil Doctoral Training Partnership Graduate Studentship BB/J014478/1(to S.A.C.), and a graduate studentship from the Tertiary Education TrustFund of Nigeria (to A.S.F.).

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