Biochemical Characterization of a Novel Bacterial Laccase and Improvement of Its Efficiency by Directed Evolution on Dye Degradation

Laccase is a copper-containing polyphenol oxidase with a wide range of substrates, possessing a good application prospect in wastewater treatment and dye degradation. The purpose of this research is to study the degradation of various industrial dyes by recombinant laccase rlac1338 and the mutant enzyme lac2-9 with the highest enzyme activity after modification by error-prone PCR. Four enzyme activities improved mutant enzymes were obtained through preliminary screening and rescreening, of which lac2-9 has the highest enzyme activity. There are four mutation sites, including V281A, V281A, P309L, S318G, and D232V. The results showed that the expression of the optimized mutant enzyme also increased by 22 ± 2% compared to the unoptimized enzyme and the optimal reaction temperature of the mutant enzyme lac2-9 was 5°C higher than that of the rlac1338, and the optimal pH increased by 0.5 units. The thermal stability and pH stability of mutant enzyme lac2-9 were also improved. With ABTS as the substrate, the kcat/Km of rlac1338 and mutant strain lac2-9 are the largest than other substrates, 0.1638 and 0.618 s–1M–1, respectively, indicating that ABTS is the most suitable substrate for the recombinant enzyme and mutant enzyme. In addition, the Km of the mutant strain lac2-9 (76 μM) was significantly lower, but the kcat/Km (0.618 s–1M–1) was significantly higher, and the specific enzyme activity (79.8 U/mg) increased by 3.5 times compared with the recombinant laccase (22.8 U/mg). The dye degradation results showed that the use of rlac1338 and lac2-9 alone had no degradation effect on the industrial dyes [indigo, amaranth, bromophenol blue, acid violet 7, Congo red, coomassie brilliant blue (G250)], however, adding small molecular mediators Ca2+ and ABTS at the same time can significantly improve the degradation ability. Compared to the rlac1338, the degradation rates with the simultaneous addition of Ca2+ and ABTS of mutant enzyme lac2-9 for acid violet 7, bromophenol blue and coomassie brilliant blue significantly improved by 8.3; 3.4 and 3.4 times. Therefore, the results indicated that the error-prone PCR was a feasible method to improve the degradation activity of laccase for environmental pollutants, which provided a basis for the application of laccase on dye degradation and other environmental pollutants.

Sono-assisted adsorption of acid violet 7 and basic violet 10 dyes from aqueous solutions: Evaluation of isotherm and kinetic parameters

In this study, the removal of acid violet 7 (AV7) and basic violet 10 (BV10) synthetic dyes was investigated using fly ash alone, ultrasound (40 kHz) alone, and combined ultrasound/fly ash with various experimental parameters such as fly ash dose, contact time, and initial concentration of dye. The adsorption capacity of the ultrasound/fly ash process increased from 5.10 to 7.43 mg g-1 for AV7, and increased from 5.16 to 7.51 mg g-1 for BV10 compared with using fly ash alone. The sono–assisted adsorption process was successful in improving the dye uptake capacity with cavitation bubbles and acoustic waves, and thus AV7 and BV10 were removed with a shorter contact time and lower fly ash dose. Obtained regeneration and reuse experiment results showed that the fly ash could be reused for four consecutive cycles of the sono–assisted adsorption process, while fly ash could be reused for two consecutive cycles of the adsorption process. The adsorption kinetics for AV7 and BV10 onto fly ash fitted Lagergren’s first–order adsorption kinetic model well. The Langmuir isotherm best described the adsorption with fly ash alone and ultrasound/fly ash process for AV7 and BV10.

Adsorption of Dyes in Aqueous Medium Using RHA and CFA

The preparation of adsorbent from the mixture of rice husk ash (RHA) and coal fly ash (CFA) has been investigated for adsorption of acid violet 7 (AV7) and brilliant green (BG) dyes. The RHA-CFA adsorbents were prepared using three different methods, i.e. reflux, magnetic co-precipitation, and magnetic template. Five different additives were used in reflux method. The results showed that RHA-CFA adsorbent prepared through reflux methods using NaOH and Na2CO3 shows higher dyes adsorption removal as compared to other methods. From zeta potential analysis, the electric charge of the outer layer of prepared adsorbent shows no effect towards adsorption of AV7 and BG dyes. By using a 3-factor, 3-level factorial design, the relationship between all variables was studied. From the response surface models, the optimum adsorbent preparation variables could be obtained by using RHA-CFA adsorbent prepared by refluxing 3:1 ratio of RHA to CFA in 1.21 M NaOH solution. The results indicated that the optimized values agree reasonably well with the validated experimental results.