Fabrication of silica on chitin in ambient conditions using silicatein fused with a chitin-binding domain.

High temperatures, harsh pH conditions, and toxic chemicals involved in the conventional synthesis and coating of silica limit the fabrication of new-generation hybrid materials immobilizing live cells and biomolecules such as enzymes and drugs. This hinders the application of inorganic-organic biohybrid materials in various fields, including bioelectronics, energy generation, and biomedicine. Silicatein, an enzyme found in siliceous sponges, catalyzes the polymerization of silica under mild conditions, that is, at room temperature and neutral pH. Silicatein was fused with a chitin-binding domain (ChBD) to selectively bind the fusion silicatein on the chitin material and with a small soluble tag called InakC, a hydrophilic protein from Pseudomonas syringae, to control the unfavorable aggregation of silicatein. The fusion silicatein was soluble in aqueous media and was successfully found to be adsorbed on the chitin material. The immobilized fusion silicatein acted as an interfacial catalyst to fabricate silica on chitin under ambient conditions. This technique can be used to fabricate inorganic-organic hybrid materials to immobilize biomolecules and can be applied to develop novel biocatalytic systems, biosensors, and tissue culture scaffolds.

Biological Route to Fabricate Silica on Cellulose Using Immobilized Silicatein Fused with a Carbohydrate-Binding Module.

Silicatein is an enzyme capable of catalyzing silica formation under mild conditions and is a promising catalyst for the fabrication of biohybrid materials. However, unfavorable aggregation of silicatein makes it unsuitable for use in material fabrication. In this study, a soluble protein tag (ProS2) and a carbohydrate-binding module (CBM) were used to develop a soluble and cellulose-binding fusion silicatein, ProS2-Sil-CBM, which can be efficiently immobilized on cellulose to form silica on it. ProS2-Sil-CBM was soluble in aqueous media and strongly bound to cellulose. ProS2-Sil-CBM bound on cellulose catalyzed the formation of a silica layer on the cellulose in the presence of tetraethyl orthosilicate as the substrate. Scanning electron microscopy (SEM) and surface elemental analysis confirmed the formation of silica on cellulose. This technique can be used to fabricate inorganic-organic hybrid materials to immobilize biomolecules and can be applied to develop novel biocatalytic systems, biosensors, and tissue culture scaffolds.

Interfacial biosilica coating of chitosan gel using fusion silicatein to fabricate robust hybrid material for biomolecular applications.

An enzyme-encapsulated silica-based hybrid material was developed using a chitosan gel. Fusion silicatein (InaKC-ChBD-Sil), silicatein fused with a soluble tag and chitin-binding domain, was employed as an interfacial catalyst to form silica on a chitosan gel matrix under physiological conditions, and horseradish peroxidase was immobilised on the hybrid material. Silica formation on the gel was verified via fluorescence microscopy using a designed fusion protein called TBP-mCherry, a fluorescent protein fused with a silica-binding peptide. We report a chitosan gel-silica hybrid material capable of encapsulating enzymes for biomedical and environmental applications.

Functional modification of mussel adhesive protein to control solubility and adhesion property.

Marine mussels produce strong underwater adhesives called mussel adhesive proteins (MAPs) that can adhere to a variety of surfaces under physiological conditions. Thus, MAPs have been investigated as a potentially sustainable alternative to conventional petrochemical-based adhesives. Recombinant MAPs would be promising for large-scale production and commercialization; however, MAPs are intrinsically adhesive, aggregative, and insoluble in water. In this study, we have developed a solubilization method for the control of MAP adhesion by fusion protein technique. Foot protein 1 (Fp1), a kind of MAP, was fused with the highly water-soluble protein, which is the C-terminal domain of ice-nucleation protein K (InaKC), separated by a protease cleaving site. The fusion protein exhibited low adhesion but high solubility and stability. Notably, Fp1 recovered its adhesive property after removal from the InaKC moiety by protease cleaving, which was evaluated and confirmed by the agglomeration of magnetite particles in water. The ability to control adhesion and agglomeration makes MAPs favorable prospects for bio-based adhesives.

Solubilization and aggregation control of silica-polymerizing enzyme fused with a removable soluble protein.

Silicatein, a silica-polymerizing enzyme, is an attractive and promising biocatalyst in many applications such as the synthesis of bio-functionalized inorganic materials under mild conditions. However, its unfavorable aggregation in aqueous media due to its intermolecular hydrophobic interactions causes difficulties in handling and applications. This study aimed to enhance the solubility of silicatein via fusion with a small soluble protein, ProS2. ProS2-Sil showed high solubility and stability in aqueous media for more than 24 h. The aggregation property of ProS2-silicatein fusion protein (ProS2-Sil) was investigated with and without cleavage of ProS2 tag by site-specific protease. When ProS2 tag was removed, silicatein became aggregated, which was analyzed by transmission electron microscope and fluorescence microscope. ProS2-Sil and mature silicatein showed similar activities in silica polymerization. The present approach allows the utilization of silicatein in the fabrication of novel and functional inorganic biohybrid materials.