Structural Insights into 2,2′-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS)-Mediated Degradation of Reactive Blue 21 by Engineered Cyathus bulleri Laccase and Characterization of Degradation Products

Abstract

Advanced oxidation processes are currently used for the treatment of different reactive dyes which involve use of toxic catalysts. Peroxidases are reported to be effective on such dyes and require hydrogen peroxide and/or metal ions. Cyathus bulleri laccase, expressed in Pichia pastoris, catalyzes efficient degradation (78 to 85%) of reactive azo dyes (reactive black 5, reactive orange 16, and reactive red 198) in the presence of synthetic mediator ABTS [2,2=-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. This laccase was engineered to degrade effectively reactive blue 21 (RB21), a phthalocyanine dye reported to be decolorized only by peroxidases. The 816-bp segment (toward the C terminus) of the lcc gene was subjected to random mutagenesis and enzyme variants (Lcc35, Lcc61, and Lcc62) were selected based on increased ABTS oxidizing ability. Around 78 to 95% decolorization of RB21 was observed with the ABTS-supplemented Lcc variants in 30 min. Analysis of the degradation products by mass spectrometry indicated the formation of several low-molecular-weight compounds. Mapping the mutations on the modeled structure implicated residues both near and far from the T1 Cu site that affected the catalytic efficiency of the mutant enzymes on ABTS and, in turn, the rate of oxidation of RB21. Several inactive clones were also mapped. The importance of geometry as well as electronic changes on the reactivity of laccases was indicated.

Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

“…This domain not only contains the mononuclear copper centre but also contributes to the formation of the binding site of the TNC. Mutations in the specific residues that compose the novel binding cavity may contribute to changes at the catalytic centre of CotA; this was also reported for various mutant sites located in domain 3, although these sites were distant from the copper centres (Gupta & Farinas, 2010;Bao et al, 2013;Kenzom et al, 2014;Jia et al, 2014).…”

Evaluation of cellobiose dehydrogenase and laccase containing culture fluids of Termitomyces sp. OE147 for degradation of Reactive blue 21

“…For this, the untreated and the treated dye samples were resolved using F 254 silica plates and 80% methanol:20% water as the mobile phase as reported [24]. Plates were dried and observed under UV at 254 nm.…”

Simplified high-throughput screening of AOX1-expressed laccase enzyme in Pichia pastoris

“…Several inactive clones were also mapped. The importance of geometry as well as electronic changes on the reactivity of laccases was indicated [8]. This high throughput screening and selection method enabled us to isolate laccase mutants with enhanced activity in a shorter period of time and is less labour intensive.…”