Extra disulfide and ionic salt bridge improves the thermostability of lignin peroxidase H8 under acidic condition.

The development of a lignin peroxidase (LiP) that is thermostable even under acidic pH conditions is a main issue for efficient enzymatic lignin degradation due to reduced repolymerization of free phenolic products at acidic pH (< 3). Native LiP under mild conditions (half-life (t1/2) of 8.2 days at pH 6) exhibits a marked decline in thermostability under acidic conditions (t1/2 of only 14 min at pH 2.5). Thus, improving the thermostability of LiP in acidic environments is required for effective lignin depolymerization in practical applications. Here, we show the improved thermostability of a synthetic LiPH8 variant (S49C/A67C/H239E, PDB: 6ISS) capable of strengthening the helix-loop interactions under acidic conditions. This variant retained excellent thermostability at pH 2.5 with a 10-fold increase in t1/2 (2.52 h at 25 °C) compared with that of the native enzyme. X-ray crystallography analysis showed that the recombinant LiPH8 variant is the only unique lignin peroxidase containing five disulfide bridges, and the helix-loop interactions of the synthetic disulfide bridge and ionic salt bridge in its structure are responsible for stabilizing the Ca2+-binding region and heme environment, resulting in an increase in overall structural resistance against acidic conditions. Our work will allow the design of biocatalysts for ligninolytic enzyme engineering and for efficient biocatalytic degradation of plant biomass in lignocellulose biorefineries.

Recombinant lignin peroxidase-catalyzed decolorization of melanin using in-situ generated H2O2 for application in whitening cosmetics.

Lignin peroxidase has high potential as ingredient in skin whitening cosmetics due to its high redox potential to oxidize recalcitrant melanin. Currently crude mixtures of lignin peroxidase from fungal fermentation are usually applied to cosmetics due to the intrinsic difficulties of expression and purification. However, the present study focused on heterologous expression and purification of lignin peroxidase isozyme H8 (LiPH8) from Phanerochaete chrysosporium and was further used for melanin decolorization. Results revealed that the optimum pH for melanin decolorization using LiPH8 was obtained at pH 4.0. The intermittent feeding of hydrogen peroxide (H2O2) was effectively elevating melanin decolorization efficiency up to 73%, since excessive H2O2 inactivated LiPH8. For cosmetic application, intermittent feeding of H2O2 is not feasible, thus glucose oxidase (GOx) from Aspergillus niger was employed for in-situ generation of H2O2. By optimizing the GOx and glucose concentrations, a melanin decolorization efficiency up to 63.3 ±â€¯2.4% was obtained within 1 h and continued to 84.0 ±â€¯1.8% in 8 h. Conclusively, lignin peroxidase-catalyzed decolorization of melanin with in-situ generated H2O2 revealed a promising approach for whitening cosmetics applications.