Application of Hypothetical Cathepsin-like Protein from Nematostella vectensis and Its Mutant Silicatein-like Cathepsin
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Application of Hypothetical Cathepsin-like Protein from Nematostella vectensis and Its Mutant Silicatein-like Cathepsin for Biosilica Production Mi-Ran Ki, Ki Ha Min and Seung Pil Pack Department of Biotechnology and Bioinformatics, Korea University, 2511 Sejong-Ro, Sejong city 339-700, Korea ABSTRACT Silicatein is general catalyst for synthesis of silica structure in siliceous sponges. However, the advent of biomimetic silicification by this recombinant version is limited by its poor yield. To overcome this limitation, we employed a cathepsin L as an alternative to silicatein. Cathepsin L has high sequence identity and similarity with silicatein alpha except cysteine other than serine residues at the active site. Here, we expressed recombinant hypothetical cathepsin-like protein (CAT) from Nematostella vectensis, displaying not only protease activity but also silica condensing activity. To increase the silica forming activity, some residues including cysteine in active site were changed into silicatein conserved residues. The mutant silicatein-like cathepsin (SLC) revealed increased protein stability in comparison with that of CAT when expressed in E. coli. The silica forming activity of SLC was comparable to that of SIL. SLC produced silica particles of size less than 50 nm which were increased to 200~300 nm in the presence of a structure-directing agent, Triton X-100. Protein immobilization by SLC-mediated silicification was performed using bovine carbonic anhydrase under ambient conditions. Immobilized protein retained its enzymatic activity for a longer time and was reused up to several times. In conclusion, CAT from Nematostella vectensis was evolved to a more soluble and available biosilica forming protein that can be applied for various silica-based materials. INTRODUCTION Silicates have a wide range of industrial uses and its nontoxic and highly biocompatible characteristics. Silification is widespread in the biological world including bacteria, single-celled protists, plants, invertebrates and vertebrates wherein gigatons of silica is formed every year in the condition of undersaturated silica concentration, around neutral pH and ambient temperature [1]. Silicatein has been discovered and isolated from spicules, skeletal element of Demosponge and are general catalysts for synthesis of silica structure in siliceous sponges. Biosilicification by silicatein is safer and lower energy catalytic pathway for nanomaterial synthesis and furthermore, it can deposit non-biological metal oxide other than silica including titanium oxide, gallium oxide
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and zirconium oxide at surfaces [2, 3]. Silicatein have been expressed both in eukaryotic and prokaryotic systems, resulting in low level and inclusion body formation when expressed in E. coli [4]. Therefore the prerequisite for the utilization of this fascinating biosilicification machinery is to obtain the sufficient amount of enzymatically active recombinant protein. Silicatein has high sequence similarity with cathepsin L protein [5]. The main differences of si
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