Oxidation of Various Kraft Lignins with a Bacterial Laccase Enzyme

Modification of kraft lignin (KL), traditionally uses harsh and energy-demanding physical and chemical processes. In this study, the potential of the bacterial laccase CotA (spore coating protein A) for oxidation of KL under mild conditions was assessed. Thereby, the efficiency of CotA to oxidize both softwood and hardwood KL of varying purity at alkaline conditions was examined. For the respective type of wood, the highest oxidation activity by CotA was determined for the medium ash content softwood KL (MA_S) and the medium ash content hardwood KL (MA_H), respectively. By an up to 95% decrease in fluorescence and up to 65% in phenol content coupling of the structural lignin units was indicated. These results correlated with an increase in viscosity and molecular weight, which increased nearly 2 and 20-fold for MA_H and about 1.3 and 6.0-fold for MA_S, respectively. Thus, this study confirms that the CotA laccase can oxidize a variety of KL at alkaline conditions, while the origin and purity of KL were found to have a major impact on the efficiency of oxidation. Under the herein tested conditions, it was observed that the MA_H KL showed the highest susceptibility to CotA oxidation when compared to the other hardwood KLs and the softwood KLs. Therefore, this could be a viable method to produce sustainable resins and adhesives.

Isolation and Characterization of a Novel Laccase LacZ1 for Lignin Degradation

Lignin is a complex natural organic polymer and is one of the primary components of lignocellulose. The efficient utilization of lignocellulose is limited because it is difficult to degrade lignin. In this study, we screened a lacz1 gene fragment encoding laccase from the macro transcriptome data of a microbial consortium WSC-6, which can efficiently degrade lignocellulose. The RT-qPCR results demonstrated that the expression level of the lacz1 gene during the peak period of lignocellulose degradation by WSC-6 increased by 30.63 times compared to the initial degradation period. Phylogenetic tree analysis demonstrated that the complete lacz1 gene is derived from Bacillus sp. and encoded laccase. The corresponding protein LacZ1 was expressed and purified by Ni-chelating affinity chromatography. The optimum temperature was 75°C, the optimum pH was 4.5, and the highest enzyme activity reached 16.39 U/mg. We found that Cu 2+ was an important cofactor needed for LacZ1 to have enzyme activity. The molecular weight distribution of lignin was determined by Gel Permeation Chromatography (GPC) and changes in the lignin structure were determined by 1H Nuclear Magnetic Resonance Spectra (1H NMR). The degradation products of lignin by LacZ1 were determined by Gas Chromatography and Mass Spectrometry (GC-MS), and three lignin degradation pathways (the gentian acid pathway, benzoic acid pathway, and protocatechuic acid pathway) were proposed. This study provides insight into the degradation of lignin and new insights into high-temperature bacterial laccase. IMPORTANCE Lignin is a natural aromatic polymer that is not easily degraded, hindering the efficient use of lignocellulose-rich biomass resources, such as straw. Biodegradation is a method of decomposing lignin that has recently received increasing attention. In this study, we screened a gene encoding laccase from the lignocellulose-degrading microbial consortium WSC-6, purified the corresponding protein LacZ1, characterized the enzymatic properties of laccase LacZ1, and speculated that the degradation pathway of LacZ1 degrades lignin. This study identified a new, high-temperature bacterial laccase that can degrade lignin, providing insight into lignin degradation by this laccase.