Biodegradation of Benzopyrene Molecule Using a Recombinant Laccase from Bacillus Subtilis as a Promising Candidate for Dye-Contaminated Environment

Abstract

Environmental remediation through biological methods is one of the main concerns of nations today. Benzopyrene (BP) is the main component of polycyclic aromatic hydrocarbons, and has been frequently found in the air, surface water, soil, and sediments. BP is resistant to remediation because of their low water solubility. Laccases are one of the best materials that could be employed for degradation of BP. Bacterial laccases are a high thermostable enzyme that can endure a vast range of pH; however, due to their low redox potential and activity, they have not been studied thoroughly. In this work, site specific mutation at a site near the Cu1, which is the primary electron acceptor in laccase-catalyzed oxidation, was applied to a laccase from Bacillus subtilis to increase laccase activity. The mutated gene cotA laccase was cloned in pET22b(+) plasmid and transformed in expression host Escherichia coli BL21 (DE3). The recombinant protein was produced under optimum induction (37 °C), purified and then analyzed through SDS-PAGE method. Enzyme activity was analyzed by specific laccase substrate (ABTS). further analysis was done for enzyme properties in different temperature, pH, and salt concentration. The results showed that the recombinant enzyme had a higher activity than native enzyme in the same condition. Determining biological degradation characteristics of BP using the recombinant laccase indicated that the recombinant laccase was more stable in different conditions and had a high enzyme potential. The functionality assays of these enzymes imply that the mutant-type is more potent given its affinity with the benzo[a]pyrene as the corresponding ligand and ABTS oxidation as a substrate under the in-silico and in-vitro conditions respectively. Furthermore, the Km and Vmax of the purified enzymes were determined in line with their activities, so that the mutant-type revealed 54% decrease in Km and 5.5-fold increase in Kcat and an increase in Kcat/Km parameter. In addition, through HPLC technique, the comparison of BP degradation by the natural and mutant enzymes confirmed the superior capability of the mutant enzyme in BP removal.