Constructing a self-assembling system for improved biocatalyst immobilization

Background.

Immobilization of biocatalysts is an important part of modern research in biocatalysis. Its key factors are the yield and precision of immobilization, enzyme stabilization during turnover and mass transfer [1]. All three factors are influenced by the carrier matrix, which typically contributes to 90-98% of the total mass, more than 50% of the catalyst costs, and an unfavorable Damköhler number >3. Immobilization matrices featuring a high catalyst loading, a high mass transfer and an easy immobilization procedure are therefore incredibly useful. Bacterial S-layers are monomolecular, self-assembling, 2D crystalline, porous arrays typically composed of only one protein [2,3]. Self-assembled S-layer proteins are very stable in water and solvent systems, and heterologous proteins can be translationally fused to S-layer proteins without interference with S-layer assembly, both in vivo and in vitro. Furthermore, S-layers provide a high-precision matrix at the nanometer-scale, which confers improved folding kinetics to the fusion partner [4] and increases the shelf-life of fused enzymes [5]. Notably, engineered S-layer chimeras with incorporated novel biological functionalities self-assemble in solution, at the air-water interface and on various solid supports [3].

Hypotheses.

1. Biocatalysts such as peroxidases, peroxygenases, catalase, and pyranose oxidase can be fused to the self-assembly domain of an S-layer protein and actively expressed in this combination. 2. The S-layer self-assembly is not compromised by the fused biocatalyst, the S-layer remains stable in water and other solvent systems. 3. The porous S-layer allows fast mass transfer for a high specific biocatalyst activity and stabilizes the turnover stability of the biocatalyst.

Methods.

Biocatalytic model enzymes targeting peroxidation, oxygenation, and oxidation reactions will be fused to S-layer proteins (e.g., SgsE from Geobacillus stearothermophilus [5]). Molecular modeling will be used to study the conformation and orientation of domains to select the best pairing of biocatalyst and carrier protein and to optimize the interprotein linker [6]. The resulting fusion proteins will be recombinantly produced in E. coli and purified by column chromatography. The assembly procedure into S-layers will be studied by electron microscopy and the activity of the fused enzymes will be assayed. The PhD candidate will select and combine functionally related enzymes such as peroxidases and hydrogen peroxide-generating enzymes or peroxidases with dehydrogenases that remove generated radicals and promote depolymerization at the expense of radical-mediated re-polymerization to generate combined biocatalysts with synergistic properties. Biocatalytic reactions on the S-layer will be investigated by scanning electrochemical microscopy [7]. The S-layers will be immobilized on ultrafiltration membranes as a structural support for testing in biocatalytic processes and biosensors. Biocatalytically active S-layers will be investigated by structural methods (e.g., cryogenic electron microscopy), kinetic methods and mass transfer measurements.

REFERENCES
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