Protein libraries were displayed on the spore coat of Bacillus subtilis, and this method was demonstrated as a tool for directed evolution under extreme conditions. Escherichia coli, yeast, and phage display suffer from protein folding, and viability issues. On the other hand, spores avoid folding concerns by the natural sporulation process, and they remain viable under harsh chemical and physical environments. The naturally occurring B. subtilis spore coat protein, CotA, was evolved for improved activity under conditions of high organic solvent concentrations. CotA is a laccase, which is a copper-containing oxidase enzyme. A CotA library was expressed on the spore coat, and ∼3000 clones were screened at 60% dimethyl sulfoxide (DMSO). A Thr480Ala variant (Thr480Ala-CotA) was identified that was 2.38-fold more active than the wild-type CotA. In addition, Thr480Ala-CotA was more active with different concentrations of DMSO ranging from 0 to 70%. The mutant was also found to be more active compared with the wild-type CotA in different concentrations of methanol, ethanol, and acetonitrile.
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