Contamination of wastewater by synthetic and natural estrogens threatens aquatic biodiversity. Laccase is an enzyme secreted by some fungi and bacteria that is capable of remediating estrogenic pollutants. To date, most bioremediation applications have utilized fungal laccases that are free in solution. While laccase immobilized on a surface has shown increased efficiency in other applications, this advantage has not been exploited in bioremediation. In recent reports, laccase from the bacterium Streptomyces coelicolor (SLAC) has exhibited unprecedented stability and activity, yet has not been tested against estrogenic pollutants. In this study, we take the initial steps toward the design of a bioremediation system using covalently-immobilized SLAC. A recombinant form of SLAC that is capable of incorporating a noncanonical amino acid (phenylalanine azide; AzF) will be attached to a surface alkyne via click chemistry. In order to build this system, a plasmid containing SLAC-AzF was co-transformed into <i>E. coli</i> host cells with another plasmid containing the essential tRNA synthetase. SLAC-AzF was overexpressed and purified using Ni-NTA affinity chromatography. Mass spectral analysis for incubations of estrogens in the presence of SLAC-AzF exhibit a variety of soluble, oxidized products. In-gel fluorescence indicates successful incorporation of the phenylalanine azide (AzF) and the ability of SLAC-AzF undergo cycloaddition via click chemistry. These results enable the development of an effective enzymatic system for bioremediation.

Preparation of small laccase (SLAC) for immobilization via click chemistry and bioremediation applications

255th National Meeting (2018)

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