Development of SpyTag/SpyCatcher-driven thermostable cyclic laccase and its application in lignin and dye degradation and polymerization of phenolic compounds.

Laccases, belonging to the superfamily of multicopper oxidases, can perform electron oxidation on a broad range of substrates, releasing only water as a by-product. Although instability and aggregation significantly constrain the industrial use of these eco-friendly biocatalysts, it is a daunting challenge for current engineering strategies to elevate these crucial enzymatic characteristics simultaneously. Here, we developed a cyclizing laccase (CyLacc) using SpyTag/SpyCatcher technology, which endows the enzyme with high thermostability and high solubility. Differential scanning calorimetry sheds light on the thermal unfolding and refolding processes of CyLacc, indicating its increased tolerance to high-temperature environments. Furthermore, utilizing large-scale molecular dynamics simulations, we found that the reduction of the hydrophobic surface and the increased flexibility of the loops are the main reasons for the increase in solubility. Compared with the wild-type laccase, the improved features led to a much higher alkaline lignin, Malachite Green, and Neutral Red decolorization efficiency of CyLacc. Furthermore, CyLacc significantly enhanced the polymerization yields of catechol and hydroquinone, from 49% and 63.6% (with wild-type Lacc) to 78.5% and 90.3%, respectively. The number-average molecular weights of the polyphenols ranged from 1,000 to 1,200 D (corresponding to the degree of polymerization that varied from 10 to 13), showing selective polymerization of phenolic compounds catalyzed by laccase. The present work, combining theory with experiment, opens up a new avenue for industrially important laccase engineering and is expected to contribute to lignin valorization and environmental protection.