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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.

Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition.

Due to their low substrate specificity, fungal laccases have a great potential in industrial applications, including the bioremediation of colored wastewaters from textile industry. However, the presence of halides in these effluents (up to 1M NaCl) which inhibit laccases is a drawback for bioremediation processes. In order to develop an efficient enzymatic remediation process for textile dye effluent, the possibility to reduce this halide inhibition is conditioned by a better understanding of the phenomenon. The present study gives a detailed account of the kinetics of chloride inhibition of both ABTS (a model substrate) and ABu62 (an anthraquinonic acid dye) oxidations catalyzed by Trametes versicolor laccase (LacIIIb). Chloride inhibition can be described by a mixed model for ABTS and a non-competitive model for ABu62 and both inhibitions are linear suggesting a single inhibitory site for chloride. Experiments were also conducted in presence of both substrates. An apparent activation of laccase was observed in the presence of ABu62 leading to an enhancement of the oxidation rate of ABTS. The extent of activation increased in the presence of chloride anions. Finally, for the first time to our knowledge, we evidenced that inhibition of ABTS oxidation by chloride can be reduced in the presence of ABu62., (Copyright © 2011 Elsevier Inc. All rights reserved.)