Poly(silicate)‐metabolizing silicatein in siliceous spicules and silicasomes of demosponges comprises dual enzymatic activities (silica polymerase and silica esterase)

Abstract

Silicon is the second most common element in the Earth's crust [1]; it possesses semi-metallic as well as metalloid properties. Silicon exists in nature in combination with oxygen as silicate ions or as silica; silica has no negative charge, while silicate anions carry a negative net electrical charge, which is counterbalanced by cations. Free silica ⁄ silicate is found both in the crystalline state (such as quartz) and in the amorphous state (such as opal). Silica ⁄ silicate is widely used in industry and medicine for the fabrication of poly(silicate), e.g. in amorphous glasses, ceramics, paints, adhesives, catalysts and photonic materials [2,3]. Furthermore, poly(silicate) is an important new material in nano(bio)technology [4,5]. This multidisciplinary research field is concerned with bio-and electronic engineering at nanometer, molecular and cellular levels [4]. Currently, production of silica require high temperature conditions and extremes of pH [6]. Hydrated amorphous silica, e.g. in the form of opal, has superb properties such as low density and high porosity. In nature, amorphous silica can be produced by diatoms by passive deposition onto an organic matrix. Siliceous sponges (Demospongiae) have the exceptional ability to synthesize silica enzymatically via silicatein [7,8]. Based on its protein sequence, silicatein is related to the proteinases of the cathepsin class [9].Silicatein has been isolated from a number of siliceous sponges, e.g. Tethya aurantium or Suberites domuncula [9,10]. If the enzyme is isolated from the skeletal elements of these animals, the spicules, it can be used in vitro to catalyze polycondensation of a wide variety of alkoxides, as well as ionic and organometallic Siliceous sponges can synthesize poly(silicate) for their spicules enzymatically using silicatein. We found that silicatein exists in silica-filled cell organelles (silicasomes) that transport the enzyme to the spicules. We show for the first time that recombinant silicatein acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization ⁄ polycondensation of silicic acid follows a distinct course. In addition, we show that silicatein cleaves the ester-like bond in bis(p-aminophenoxy)-dimethylsilane. Enzymatic parameters for silica esterase activity are given. The reaction is completely blocked by sodium hexafluorosilicate and E-64. We consider that the dual function of silicatein (silica polymerase and silica esterase) will be useful for the rational synthesis of structured new silica biomaterials.

Self‐healing, an intrinsic property of biomineralization processes

“…After having identified the major protein within the spicules as silicatein, our group succeeded to show that silicatein is a true enzyme with all characteristics known for other enzymes (40,41). In general, enzymes are defined as (i) three-dimensionally structured proteins that have the same structure at the start and at the end of the reaction; (ii) to be substratespecific, implying that each enzyme is optimized for a particular reaction transition state, (iii) to lower the free energy of thermodynamics to overcome the transition states, (iv) to be characterized by a defined Michaelis-Menten constant (K m ), reflecting the binding affinity to the substrate, (v) to show a maximum velocity (v max ) and (vi) to be inhibitable (ref.…”

“…The turnover value (molecules of converted substrate per enzyme molecule per second) for silicatein in the silica esterase assay is 5.2; in comparison, the rate for the human cathepsin L enzyme is with 20, slightly higher (44). The specificity of the silica-metabolizing reaction was determined by showing that a replacement of silicatein in the assays by bovine serum albumin resulted in no measurable silica synthesis (40). The pH value of the enzyme-driven biopolycondensation reactions was kept at 7.4/7.5 (40,41) and not varied, considering the fact that the autocatalytic polycondensation reaction is strongly dependent on small changes of the pH (45).…”

“…The key enzyme kinetic parameters of the enzyme-catalyzed polycondensation reaction have been determined (40,41). For the enzyme assays, either native silicatein or recombinant protein has been used at adequate final concentration of around 10 lg/mL in the reaction mixture.…”

“…The specificity of the silica-metabolizing reaction was determined by showing that a replacement of silicatein in the assays by bovine serum albumin resulted in no measurable silica synthesis (40). The pH value of the enzyme-driven biopolycondensation reactions was kept at 7.4/7.5 (40,41) and not varied, considering the fact that the autocatalytic polycondensation reaction is strongly dependent on small changes of the pH (45). The data, listed here, conclusively show that the silicatein-mediated biosilica formation is enzyme driven.…”

Analysis of silicatein gene expression and spicule formation in the demosponge Amphimedon queenslandica

“…Silica in its different forms is widely used in everyday products such as glasses, paints, adhesives, stabilizers, food additives and high-tech products Schröder et al 2007b). Industrially produced silica-based materials require extremely high pressures and temperatures, and yield significant amounts of harmful chemical waste (Morse 1999;Müller et al 2008a). In contrast, sponges have this unique ability to form these highly ordered and hierarchical structures under physiological conditions, at ambient temperature and pressure.…”