ABSTRACT
The cDNA fragment encoding the catalytic domain of the new silicatein-like cathepsin enzyme LoCath was expressed in a strain Top10 of Escherichia coli, extracted and purified via nickel-affinity chromatography. Recombinant enzyme performed silica-polymerizing activity when mixed with water-soluble silica precursor-tetrakis-(2-hydroxyethyl)-orthosilicate. Scanning electron microscopy revealed hexagonal, octahedral and ß-tridimit crystals. Energy dispersion fluorescence X-ray spectrometry analysis showed that all these crystals consist of pure silicon oxide. It is the first report about the ability of marine sponge's cathepsin to polymerize silicon, as well as about the structure and composition of the silicon oxide crystal formed by recombinant cathepsin. Further study of the catalytic activity of silicatein and cathepsin will help to understand the biosilification processes in vivo, and will create basis for biotechnological use of recombinant proteins for silicon polymerization.
Subject(s)
Cathepsins/metabolism , Porifera/enzymology , Silicon/chemistry , Animals , Microscopy, Electron, Scanning , Polymerization , Recombinant Proteins/metabolismABSTRACT
We suggested to use tetrakis(2-hydroxyethyl)orthosilicate (THEOS) as a substrate for silicateins--an enzyme family playing a key role in formation of skeleton in marine sponges. We compared THEOS with tetraethylorthosilicate (TEOS)--a commonly used substrate for silicateins. These substrates were tested in reaction of amorphous silica formation in vitro catalyzed by silicatein Al from sponge Latrunculia oparinae. It was found that reaction with THEOS occurs more efficiently than with TEOS, probably due to high water solubility and higher hydrolysis rate of THEOS.