Biochemical and Biophysical Characterisation of Silicatein

UoM administered thesis: Phd

  • Authors:
  • Stephanie Caslin

Abstract

The use of biocatalysts in organosilicon chemistry is not widely studied. The application of a catalyst that operates under ambient conditions would offer a more sustainable method in the production of organosilicon compounds and silicon based materials, in particular silicone, which is almost universally used in many consumer products and industrial processes. Silicatein, an enzyme found exclusively in the marine sponge catalyses the polycondensation of silica and would be a good candidate for the biocatalysis of silicon containing organic compounds. Recombinant silicatein-α has been previously shown to accept simple compounds as substrates such as silicon ethoxide and TEOS1 along with more complex silanes and organosiloxanes.2 A steady and reliable supply of active, soluble protein was achieved by screening a range of protein production parameters, such as a variety of E. coli strains and fusion partners and a range of incubation temperatures. Further investigations led to the optimisation of purification and isolation methods to obtain fusion protein and native silicatein-α for structural and functional analysis. Therefore, the existing methods were refined to produce both recombinant enzymes more efficiently, reducing the overall time of protein purification. A range of enzyme variants was used to characterise and explore the functionality of each catalytic residue on the overall catalytic ability. Kinetic data showed although similar KM values were recorded the variants, the wild type enzyme has the highest catalytic efficiency in the region of 46 min-1 µM-1. Therefore, the presence of all three catalytic residues are necessary for full catalytic activity towards this substrate. TF-silicatein-α and Ser26Cys revealed no esterase and protease activity, however, Asn185Asp displayed a small level of esterase activity (1.24 % conversion). CD and DLS analysis confirmed structural integrity under the standard assay conditions with secondary structural predicted to be similar to theoretically calculated data from crystallographic data in the PDB. DLS and MALS data also highlighted two potential buffer conditions that are suitable for maintaining protein homogeneity in solution. MALS data shows silicatein-α to be monomeric with a Mw of ~ 30 kDa. Silyl etherification and trans-etherification was catalysed by TF-silicatein-α at a higher rate of conversion at 90 °C and showed preference towards S-enantiomers. In addition, preliminary data indicated the possibility of a coupled enzyme synthesis with an S-specific ADH. In summary this work confirms TF-silicatein-α as a robust, highly thermostable and enantioselective biocatalyst with potential applications in many industries that use organosilicon compounds.

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Original languageEnglish
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Award date1 Aug 2018