Laccase-Mediator System Using a Natural Mediator as a Whitening Agent for the Decolorization of Melanin.

In this study, a laccase-mediator system (LMS) using a natural mediator was developed as a whitening agent for melanin decolorization. Seven natural mediators were used to replace synthetic mediators and successfully overcome the low redox potential of laccase and limited access of melanin to the active site of laccase. The melanin decolorization activity of laccases from Trametes versicolor (lacT) and Myceliophthora thermophila (lacM) was significantly enhanced using natural mediators including acetosyringone, syringaldehyde, and acetovanillone, which showed low cytotoxicity. The methoxy and ketone groups of natural mediators play an important role in melanin decolorization. The specificity constants of lacT and lacM for melanin decolorization were enhanced by 247 and 334, respectively, when acetosyringone was used as a mediator. LMS using lacM and acetosyringone could also decolorize the melanin present in the cellulose hydrogel film, which mimics the skin condition. Furthermore, LMS could decolorize not only synthetic eumelanin analogs prepared by the oxidation of tyrosine but also natural melanin produced by melanoma cells.

Immobilization of laccase via cross-linked enzyme aggregates prepared using genipin as a natural cross-linker.

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs.

Effect of hydrogen bond donor on the choline chloride-based deep eutectic solvent-mediated extraction of lignin from pine wood.

In this work, twenty-five kinds of choline chloride (ChCl)-based deep eutectic solvents (DESs) containing acid, hydroxyl, amide, and binary hydrogen bond donors (HBDs) were prepared and successfully used to pretreat pine wood powder. As a result of the pretreatment, the glucan content in the pretreated biomass was increased, whereas the contents of hemicellulose and lignin were significantly decreased. The biomass pretreatment efficiency of the DESs had improved with increasing the polarity and hydrogen bond acidity (α) of the DESs. Among the studied DESs, ChCl:lactic acid:formic acid (1:1:1) with the highest α value was the most efficient DES in extracting lignin from biomass. The pretreated biomass also showed an enhanced enzymatic saccharification yield owing to the decreased particle size of the biomass and reduced content of hemicellulose and lignin. During the pretreatment process of biomass using DESs, the extracted lignin could be recovered successfully, with a yield of up to 60% and purity of over 90%. The molecular weight of the extracted lignin was much lower than that of the native cellulolytic enzyme lignin. The DES used for pretreatment process could be also successfully reused with high recovery yield of DES and high retention of delignification capacity.

Effect of Cellulose Solvents on the Characteristics of Cellulose/Fe2O3 Hydrogel Microspheres as Enzyme Supports.

Cellulose hydrogels are considered useful biocompatible and biodegradable materials. However, as few cellulose-dissolving solvents can be used to prepare cellulose hydrogel microspheres, the use of unmodified cellulose-based hydrogel microspheres for enzyme immobilization remains limited. Here, we prepared cellulose/Fe2O3 hydrogel microspheres as enzyme supports through sol-gel transition using a solvent-in-oil emulsion. Cellulose-dissolving solvents including 1-ethyl-3-methylimidazolium ([Emim][Ac]), an aqueous mixture of NaOH and thiourea, tetrabutylammonium hydroxide, and tetrabutylphosphonium hydroxide were used to prepare regular shaped cellulose/Fe2O3 microspheres. The solvent affected microsphere characteristics like crystallinity, hydrophobicity, surface morphology, size distribution, and swelling properties. The immobilization efficiency of the microspheres for lipase was also significantly influenced by the type of cellulose solvent used. In particular, the lipase immobilized on cellulose/Fe2O3 microspheres prepared using [Emim][Ac] showed the highest protein loading, and its specific activity was 3.1-fold higher than that of free lipase. The immobilized lipase could be simply recovered by a magnet and continuously reused.