Recombinant silicateins as model biocatalysts in PNAS PLUS 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 nosiloxanes. Several attempts have been made to use hydrolytic Porifera) is unique in nature for catalyzing the formation of enzymes such as lipases and proteases for the hydrolysis and inorganic silica structures, which the organisms incorporate into condensation of the Si–O bond (11–13). Although they clearly their skeleton. However, the synthesis of organosiloxanes cata- demonstrate the feasibility of the general concept, the range of lyzed by these enzymes has thus far remained largely unexplored. enzymes tested have so far met with only limited success in To investigate the reactivity of these enzymes in relation to this regard to synthetic yield and substrate scope. important class of compounds, their catalysis of Si–O bond hydro- In contrast, poriferans (marine sponges) that use silica as part lysis and condensation was investigated with a range of model of their inorganic skeleton use a family of enzymes termed the organosilanols and silyl ethers. The enzymes’ kinetic parameters silicateins to catalyze the polymerization of soluble silicates into were obtained by a high-throughput colorimetric assay based on silica (14–16). The primary sequences of these enzymes have the hydrolysis of 4-nitrophenyl silyl ethers. These assays showed been reported and they bear a remarkable homology with pro- k K unambiguous catalysis with cat/ m values on the order of teases of the cathepsin family, with ∼65% sequence similarities – −1 μ −1 2 50 min M . Condensation reactions were also demonstrated and ∼50% sequence identities relative to cathepsin L. Both en- by the generation of silyl ethers from their corresponding silanols zymes share a similar Xaa–His–Asn catalytic triad at their active CHEMISTRY and alcohols. Notably, when presented with a substrate bearing site, although in the silicateins a Ser residue occupies the Xaa both aliphatic and aromatic hydroxy groups the enzyme preferen- position rather than Cys in cathepsin L. Previous reports have tially silylates the latter group, in clear contrast to nonenzymatic shown that silicatein-α (Silα), the prototypical member of this silylations. Furthermore, the silicateins are able to catalyze trans- etherifications, where the silyl group from one silyl ether may be family, can catalyze the hydrolysis of ethoxysilanes such as tet- transferred to a recipient alcohol. Despite close sequence homol- raethoxysilane (TEOS) and triethoxyphenylsilane (17). Because the silicateins have evolved specifically to manipulate ogy to the protease cathepsin L, the silicateins seem to exhibit no – significant protease or esterase activity when tested against anal- the Si O bond, these enzymes may offer a better starting point ogous substrates. Overall, these results suggest the silicateins are for further elaboration into practical biocatalysts in organo- BIOCHEMISTRY promising candidates for future elaboration into efficient and se- siloxane chemistry. This paper outlines the performance of het- α lective biocatalysts for organosiloxane chemistry. erologously produced Sil for both the hydrolysis and condensation of a range of model organosiloxanes. In the process, the devel- silicatein | biocatalysis | organosilicon | organosiloxane | silyl ether opment of a colorimetric high-throughput screening method for silyl ether bond hydrolysis based on the 4-nitrophenoxylate he organosiloxanes, compounds containing C–Si–O moieties, Trepresent a class of compounds with a truly diverse range of Significance applications. They are commonly used in the form of poly- siloxane “silicone” polymers as components of industrial and Organosiloxanes are components in a huge variety of con- consumer products for a variety of purposes such as bulking sumer products and play a major role in the synthesis of fine agents, separation media, protective coatings, lubricants, emul- chemicals. However, their synthetic manipulation primarily sifiers, and adhesives (1–3). Their use as auxiliaries in the relies on the use of chlorosilanes, which are energy-intensive to chemical synthesis of complex molecules is also long-established produce and environmentally undesirable. Synthetic routes (4–6). However, the production and synthetic manipulation of that operate under ambient conditions and circumvent the these compounds are almost entirely dependent on chlorosilane need for chlorinated feedstocks would therefore offer a more feedstocks, which are environmentally undesirable and energy- sustainable route for producing this class of compounds. Here, intensive to produce (7, 8). Furthermore, organosiloxanes, which a systematic survey is reported for the silicatein enzyme, which are entirely anthropogenic in origin, are now known to be per- is able to catalyze the hydrolysis, condensation, and exchange sistent environmental contaminants because little attempt is of the silicon–oxygen bond in a variety of organosiloxanes made to recover and recycle them (3). Synthetic routes that use, under environmentally benign conditions. These results sug- and ultimately recycle, siloxanes and silanols as alternatives gest that silicatein is a promising candidate for development of would in principle be more environmentally sound. selective and efficient biocatalysts for organosiloxane chemistry. One possible strategy toward improved sustainability is to harness enzymes for chemical processing. Such biocatalysts are 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; attractive because they offer highly efficient synthesis in terms of and S.Y.T.D., S.A.C., P.Q., S.P.d.V., and L.S.W. wrote the paper. yields and regio- and stereospecificity, together with an ability to The authors declare no conflict of interest. promote reactions under mild conditions and a minimal reliance This article is a PNAS Direct Submission. on halogenated or metallic feedstocks (9, 10). The use of en- Freely available online through the PNAS open access option. – zymes to manipulate the Si O bond would therefore potentially 1To whom correspondence should be addressed. Email: [email protected]. offer more sustainable routes to the synthesis of many com- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pounds, as well as for the eventual recycling and reuse of orga- 1073/pnas.1613320114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1613320114 PNAS Early Edition | 1of7 Downloaded by guest on September 29, 2021 chromophore is also reported. Additionally, the protease and es- 16000 α terase activity of Sil against analogous substrates is described. 12000 ) Results and Discussion -1 8000 Production and Characterization of Silα. dmol To acquire this enzyme, a 2 4000 TF-Silα synthetic vector containing cDNA encoding for the mature wild- 0 type Silα from Suberites domencula, fused to an N-terminal (° cm TF-Silα (Mutant-Ser26Ala) hexahistidine tag and codon optimized for expression in Escherichia ME -4000 coli, was used. It is known that the mature form of the protein is -8000 highly hydrophobic and difficult to produce in soluble form (16, 18). -12000 Thus, in attempts to improve its solubility the gene was also subcl- 190 210 230 250 oned with the sequences for a number of proteins known to enhance Wavelength (nm) solubility and folding. Sequences encoding for GST, thioredoxin, small ubiquitin-like modifier, maltose binding protein, or trigger Fig. 2. CD spectra plots of molar ellipticity against wavelength for TF-Silα factor (TF) were inserted between the hexahistidine tag and Silα and TF-Silα(Ser26Ala). and the genes transformed into a variety of E. coli BL21(DE3) strains including Arctic-Express and Origami. Expression trials In contrast, Silα could be maintained in a homogeneous state were then carried out by varying the induction conditions, includ- only at dilute concentrations (micromolar range), which were ing the concentration of the induction agent (isopropyl β-D-1- insufficient for CD measurements. However, analysis by non- thiogalactopyranoside), incubation temperature, and incubation time. denaturing gel electrophoresis for Silα clearly showed a single Overall, these optimization experiments showed that all of the band, indicating nonaggregation (SI Appendix, Fig. S1B). The candidate proteins expressed well in E. coli but only the TF-Silα α protein advanced through the gel at a similar rate as the 25 kDa fusion gave the protein in soluble form. As expected, the Sil reference protein, suggesting that it was of approximately similar (without any fusion tag) was almost entirely insoluble. However, size, globular, and monomeric. it was found that addition of the nondenaturing detergents Tri- ton X-100 and CHAPS into the lysis buffer enabled the recovery Determination of Enzymatic