Self-Initiated Co-Immobilization Of Laccase-Mediator System And The Evaluation For Its Enzymatic Conversion Of Organic Dyes

Laccase is an environmental-friendly biocatalyst that can oxidize various organic pollutants with the concomitant reduction of oxygen to water. However, its practical application is far less than satisfactory due to the low stability and poor reusability of free laccase. A highly stable and efficient biocatalyst (denoted as Immo-Lac) was fabricated by encapsulating Trametes versicolor laccase within a chitosan grafted polyacrylamide (CTS-g-PAM) hydrogel through free radical polymerization. Scanning electron microscopy and nitrogen adsorption-desorption tests demonstrate that channels of diameter of 10-20 μm were regularly distributed throughout the sponge-like Immo-Lac. Besides, there were massive mesopores in the lamellar walls of the hydrogel. The recovered activity of Immo-Lac was 40.8%. As compared with the free laccase, the Immo-Lac showed enhanced thermal and chemical stability. The positive surface charge of the Immo-Lac endowed it with a pre-enrichment effect in the treatment of anionic dyes. In a continuous cycle batch decoloration of malachite green, the Immo-Lac showed much better durability than the free laccase.The addition of redox mediator was necessary for the catalytic oxidation of organic pollutants by laccase. Given the high cost and the potential toxicity of these dissolved mediators, how to recover and reuse them is an attractive but challenging task. To address the bottlenecks in laccase-based catalysis, a novel biocatalyst (Immo-LMS) was fabricated by simultaneously immobilizing both laccase and a mediator (acetylacetone, abbreviated as AA) into CTS-g-PAM hydrogel through the laccase-AA initiated polymerization. This self-initiated immobilization process avoided the forced conformational change of laccase in the passive embedding to pre-existing carriers. The Immo-LMS had a sponge-like structure and a recovered enzyme activity of 32.0%. Both nuclear magnetic resonance and cyclic voltammetry experiments confirm that AA after covalently bonded to the polymer chain via the centered carbon still possessed redox ability and could enhance the catalytic oxidation as a mediator. The Immo-LMS had the highest substrate conversion quantity (6.94 μmol U-1) to malachite green, followed by the sole immobilized laccase (4.64 μmol U-1) and the immobilized laccase with an external mediator (1-hydroxybenzotriazole, HBT) (3.25 μmol U-1). The good performance of the Immo-LMS suggests that the co-immobilization of laccase and mediator through the self-initiated one-pot process was a promising pattern for the recycle and reuse of both laccase and mediator. Such a strategy is expected to be helpful to cut down the running cost as well as the potential toxicity that come from synthetic mediators. We believe that this method is an innovative immobilization pattern in the development of immobilized laccase.