An enzymatic system for decolorization of wastewater dyes using immobilized CueO laccase-like multicopper oxidase on poly-3-hydroxybutyrate

Abstract

The presence of synthetic dyes in wastewaters generated by the textile industry constitutes a serious environmental and health problem that urges the scientific community on an appropriate action. As a proof-of-concept, we have developed a novel approach to design enzymatic bioreactors with the ability to decolorize dye solutions through the immobilization of the bacterial CueO laccase-like multicopper oxidase from Escherichia coli on polyhydroxybutyrate (PHB) beads by making use of the BioF affinity tag. The decolorization efficiency of the system was characterized by a series of parameters, namely maximum enzyme adsorption capacity, pH profile, kinetic constants, substrate range, temperature and bioreactor recycling. Depending on the tested dye, immobilization increased the catalytic activity of CueO by up to 40-fold with respect to the soluble enzyme, reaching decolorization efficiencies of 45-90%. Our results indicate that oxidase bioreactors based on polyhydroxyalkanoates are a promising alternative for the treatment of coloured industrial wastewaters.

Figure 1

Correspondence between the estimated logP for each dye and their nonspecific adsorption to PHB.

Figure 2

Decolorization of textile dyes by BioF–CueO. Samples (20 mL) were incubated in batch with PHB‐immobilized BioF–CueO by shaking at 150 r.p.m. A blank containing no enzyme or support was subtracted in all cases. Results are the mean of triplicate experiments and are shown as decrease in absorbance with respect to the initial values. The first two plots (Reactive Blue 19 and Cibacron Blue 3G‐A) correspond to dyes that partially adsorb to PHB, while the last three plots (Reactive Black 5, New Coccine and Indigo Carmine) correspond to dyes showing negligible adsorption (<5%) to PHB.

Figure 3

Examples of dye degradation with BioF–CueO after 20 h incubation. Conditions as detailed in Fig. 2.

Figure 4

Optimization of Cu²⁺ addition. An RB5 solution (1 mL) was incubated in batch on a digital rotary mixer for 1 h with PHB (approximately 0.1 mL of bed volume) functionalized with BioF–CueO as described in Experimental Procedures. After each incubation cycle, the supernatant was removed and analysed, and the resin was washed with Tris buffer and then loaded with a fresh dye solution for a subsequent decolorization cycle. Global means that 0.1 mM Cu²⁺ was maintained constant in the dye solution during the decolorization process. Only in resin means equilibration of the PHB support with 0.1 mM Cu²⁺ (0.1 mL), followed by washing and subsequent addition of dye solution without the metal ion. Results (mean of triplicates) are shown as decrease in absorbance with respect to the initial values.

Figure 5

Effect of temperature on BioF–CueO‐mediated dye degradation. Solutions of RB5 (black bars) or IC (grey bars) in 20 mM Tris buffer, pH 7.0, plus 0.1 mM CuSO ₄ were preincubated for 30 min at the temperatures shown in the graphs, then added to the functionalized resin and left to react at 25 °C for 24 h. Blanks (enzyme‐free) were subtracted in each experiment. Results (mean of triplicates) are shown as decrease in absorbance with respect to the initial values.

Figure 6

A, Decolorization of an industrial wastewater sample by immobilized BioF–CueO in the absence (black bars) or the presence (grey bars) of 0.1 mM Cu²⁺. B, Effect of pH and solution conditions on activity. Samples contained 1.7 mg of protein per 100 mg of PHB.