Performance enhancement of white rot fungi extracellular enzymes via new hydrogel microenvironments for remediation of benzo[a]pyrene contaminated soil

Organic pollutants with low solubility and high ecotoxicity, mutagenicity, and carcinogenicity, are rapidly entering and accumulating in soil, resulting in soil pollution. Several methods have been investigated for remediation of organic contaminated soil, including enzymatic remediation approach. However, free enzymes are easily deactivated, which hinders their practical application in soil remediation. Immobilization of enzyme improves its stability and catalytic performance, but the immobilized material itself becomes secondary pollutants in soil. In this study, Trametes versicolor extracellular enzyme was immobilized on the degradable calcium alginate hydrogel microspheres. The laccase maintained a high activity. In addition, the addition of cellulose improved the strength of the carrier. Hydrogel microspheres solved the problems of easy inactivation of free enzyme and secondary contamination of immobilized materials. By a novel combination of extracellular enzymes and hydrogel microenvironments, immobilized enzymes not only demonstrate outstanding performance in thermal stability and pH adaptability, but also achieves a significant improvement in biocatalytic activity for benzo[a]pyrene contaminated soil. The thermal stability of immobilized enzyme was much higher than that of free enzyme. When the temperature increased to 50 °C, the activity of immobilized enzyme remained at 93.15% of the maximum enzyme activity, while the activity of free enzyme decreased to 63.76%. At pH 8, the immobilized enzyme activity maintained 74.84% of the maximum enzyme activity, while the free enzyme activity was only 11.86%. Immobilized enzymes can effectively remove 91.40% of benzo[a]pyrene from soil within 96 h. Furthermore, the catalytic oxidation of benzo[a]pyrene by enzymes that have been immobilized ultimately results in the production of 6,12-benzo[a]pyrene-dione. Molecular dynamics simulation showed that the catalytic degradation of benzo[a]pyrene was mainly through the interaction of amino acid residues PRO-391 with the Pi-alkyl of benzo[a]pyrene. This study presents an innovative strategy for designing and developing immobilized enzymes for use in biocatalytic applications related to eco-remediation of soil.