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In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE , cotG , and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a His 6 tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be 50°C. Upon treatment with known laccase inhibitors, including EDTA, SDS, and NaN 3 , the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.

Decolorization of Acid Green 25 by Surface Display of CotA laccase on Bacillus subtilis spores