Yuji Tsutsumi

Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes

Bisphenol A (BPA) and nonylphenol (NP) were treated with manganese peroxidase (MnP) and laccase prepared from the culture of lignin-degrading fungi. Laccase in the presence of 1-hydroxybenzotriazole (HBT), the so-called laccase-mediator system, was also applied to remove the estrogenic activity. Both chemicals disappeared in the reaction mixture within a 1-h treatment with MnP but the estrogenic activities of BPA and NP still remained 40% and 60% in the reaction mixtures after a 1-h and a 3-h treatment, respectively. Extension of the treatment time to 12 h completed the removal of estrogenic activities of BPA and NP. The laccase has less ability to remove these activities than MnP, but the laccase-HBT system was able to remove the activities in 6 h. A gel permeation chromatography (GPC) analysis revealed that main reaction products of BPA and NP may be oligomers formed by the action of enzymes. Enzymatic treatments extended to 48 h did not regenerate the estrogenic activities, suggesting that the ligninolytic enzymes are effective for the removal of the estrogenic activities of BPA and NP.

The Difference of Reactivity between Syringyl Lignin and Guaiacyl Lignin in Alkaline Systems

The difference of reactivity between syringyl lignin and guaiacyl lignin in alkaline systems were investigated by using syringyl and guaiacyl types of β-aryl ether lignin model compounds, softwood and hardwood dioxane lignins, and softwood and hardwood meals. In the lignin model study, β-aryl ether of syringyl lignin model was cleaved much faster than that of guaiacyl lignin model by soda and soda/anthraquinone treatment. The formation of coniferyl alcohol and sinapyl alcohol from guaiacyl lignin model and syringyl lignin model, respectively, was almost proportional to the cleavage of β-aryl ether under these conditions. Based on the model studies, coniferyl alcohol and sinapyl alcohol from isolated lignin and protolignin by soda and soda/anthraquinone treatments were determined in order to evaluate the extent of β-aryl ether cleavage in the terminal units of lignin. The results in both the dioxane lignins study and the wood meals study also indicate that β-aryl ether of syringyl lignin is cleaved much more easily than that of guaiacyl lignin and that there is no difference of reactivity of guaiacyl lignin in hardwood lignin and softwood lignin. This high reactivity of syringyl lignin may contribute the faster delignification rate of hardwood than softwood.

Lignin dehydrogenative polymerization mechanism : a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in β-O-4 linkage

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO‐C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO‐C was capable of oxidizing high‐molecular‐weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO‐C characteristics are important to produce polymer lignin. The results suggest that CWPO‐C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.

Polyethylene degradation by lignin-degrading fungi and manganese peroxidase

Degradation of high-molecular-weight polyethylene membrane by lignin-degrading fungi, IZU-154, Phanerochaete chrysosporium, and Trametes versicolor, was investigated under various nutritional conditions. IZU-154 showed the most significant polyethylene degradation among the three lignin-degrading fungi under nitrogen- or carbon-limited culture conditions. Furthermore, for T. versicolor and P. chrysosporium, the addition of Mn(II) into nitrogen- or carbon-limited culture medium enhanced polyethylene degradation. These results suggest that polyethylene degradation is related to ligninolytic activity of lignin-degrading fungi. Treatment of polyethylene membrane with partially purified manganese peroxidase (MnP) caused significant degradation in the presence of Tween 80, Mn(II), and Mn(III) chelator. This result demonstrates that MnP is the key enzyme in polyethylene degradation by lignin-degrading fungi.

Diverse functions and reactions of class III peroxidases

Higher plants contain plant-specific peroxidases (class III peroxidase; Prxs) that exist as large multigene families. Reverse genetic studies to characterize the function of each Prx have revealed that Prxs are involved in lignification, cell elongation, stress defense and seed germination. However, the underlying mechanisms associated with plant phenotypes following genetic engineering of Prx genes are not fully understood. This is because Prxs can function as catalytic enzymes that oxidize phenolic compounds while consuming hydrogen peroxide and/or as generators of reactive oxygen species. Moreover, biochemical efforts to characterize Prxs responsible for lignin polymerization have revealed specialized activities of Prxs. In conclusion, not only spatiotemporal regulation of gene expression and protein distribution, but also differentiated oxidation properties of each Prx define the function of this class of peroxidases.

Sinapyl alcohol-specific peroxidase isoenzyme catalyzes the formation of the dehydrogenative polymer from sinapyl alcohol

Two peroxidases, CWPO-A and CWPO-C, were isolated from the cell walls of poplar (Populus alba L.) callus culture. The cationic CWPO-C showed a strong preference for sinapyl alcohol over coniferyl alcohol as substrate. Thus, the monolignol utilization of CWPO-C is unique compared with other peroxidases, including anionic CWPO-A and horseradish peroxidase (HRP). CWPO-C polymerized oligomeric sinapyl alcohol (S-oligo) and sinapyl alcohol, producing a polymer of greater molecular weight. In contrast, HRP, which is specific to coniferyl alcohol, produced sinapyl alcohol dimers, rather than catalyzing polymerization. Adding coniferyl alcohol as a radical mediator in the HRP-mediated reaction did not result in S-oligo polymerization. This report shows that CWPO-C is an isoenzyme specific to sinapyl alcohol that polymerizes oligomeric lignols. Its catalytic activity toward oligomeric lignols may be related to the lignification of angiosperm woody plant cell walls.

Putative cationic cell-wall-bound peroxidase homologues in arabidopsis, AtPrx2, AtPrx25, and AtPrx71, are involved in lignification

The final step of lignin biosynthesis, which is catalyzed by a plant peroxidase, is the oxidative coupling of the monolignols to growing lignin polymers. Cationic cell-wall-bound peroxidase (CWPO-C) from poplar callus is a unique enzyme that has oxidative activity for both monolignols and synthetic lignin polymers. This study shows that putative CWPO-C homologues in Arabidopsis , AtPrx2, AtPrx25, and AtPrx71, are involved in lignin biosynthesis. Analysis of stem tissue using the acetyl bromide method and derivatization followed by the reductive cleavage method revealed a significant decrease in the total lignin content of ATPRX2 and ATPRX25 deficient mutants and altered lignin structures in ATPRX2, ATPRX25, and ATPRX71 deficient mutants. Among Arabidopsis peroxidases, AtPrx2 and AtPrx25 conserve a tyrosine residue on the protein surface, and this tyrosine may act as a substrate oxidation site as in the case of CWPO-C. AtPrx71 has the highest amino acid identity with CWPO-C. The results suggest a role for CWPO-C and CWPO-C-like peroxidases in the lignification of vascular plant cell walls.

Substrate-Specific Peroxidases in Woody Angiosperms and Gymnosperms Participate in Regulating the Dehydrogenative Polymerization of Syringyl and Guaiacyl Type Lignins

Substrate specificities of enzymes involved in monolignol biosynthesis are recognized to control differentiation of guaiacyl and syringyl lignin, but peroxidases are regarded to have rather less substrate specificity and their specificity related to lignification has not been fully elucidated. We have investigated the substrate specificity of peroxidases (EC1.11.1.7) in poplar (Populus alba L.) and Japanese cedar (Cryptomeria japonica D. Don) with respect to the dehydrogenative polymerization of monolignols. Peroxidases were fractionated into three groups, namely soluble peroxidases ionically bound peroxidases (IPO). and covalently bound peroxidases. Populus IPO was found to have the largest preference for sinapyl alcohol among all of peroxidases from both woody plant species, and only this peroxidase could produce the dehydrogenative polymer (DHP) from sinapyl alcohol. On the other hand. all peroxidases from both Cryptomeria and Populus easily produced the DHP from coniferyl alcohol. The results support that the participation of substrate-specific peroxidases is an important factor regulating the accumulation of syringyl and guaiacyl lignins in angiosperms and gymnosperms. Electrophoresis of IPO revealed the existence of syringyl-specific isoperoxidase only in Populus IPO.

The cationic cell-wall-peroxidase having oxidation ability for polymeric substrate participates in the late stage of lignification of Populus alba L

Previously we reported that purified Cell Wall Peroxidase-Cationic (CWPO-C) from poplar callus (Populus alba L.) oxidizes sinapyl alcohol and polymeric substrate unlike other plant peroxidases and proposed that this isoenzyme is a conceivable lignification specific peroxidase. In this study, we cloned full-length cDNA of CWPO-C and investigated the transcription of CWPO-C gene in various organs and the localization of CWPO-C protein in the differentiating xylem of poplar stem.Real-time PCR analyses indicated that CWPO-C gene is constitutively expressed in the developing xylem, leaf, and shoot but not affected by many stress treatments. Immunohistochemical analysis showed that CWPO-C locates in the middle lamellae, cell corners, and secondary cell walls of the fiber cells during the lignification. The intensity of the CWPO-C labeling increased gradually from the cell wall thickening stage to mature stage of fiber cells, which is very consistent with the increase of lignin content in the developing xylem. These results strongly support that CWPO-C is responsible for the lignification of the secondary xylem. Interestingly, immuno-labeling of CWPO-C was also observed inside of the ray parenchyma cells instead no signals were detected within the developing fiber cells. This suggests that CWPO-C is biosynthesized in the parenchyma cells and provided to the middle lamellae, the cell corners, and the cell walls to achieve lignin polymerization.

Simultaneously disrupting AtPrx2, AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem

Plant class III heme peroxidases catalyze lignin polymerization. Previous reports have shown that at least three Arabidopsis thaliana peroxidases, AtPrx2, AtPrx25 and AtPrx71, are involved in stem lignification using T-DNA insertion mutants, atprx2, atprx25, and atprx71. Here, we generated three double mutants, atprx2/atprx25, atprx2/atprx71, and atprx25/atprx71, and investigated the impact of the simultaneous deficiency of these peroxidases on lignins and plant growth. Stem tissue analysis using the acetyl bromide method and derivatization followed by reductive cleavage revealed improved lignin characteristics, such as lowered lignin content and increased arylglycerol-β-aryl (β-O-4) linkage type, especially β-O-4 linked syringyl units, in lignin, supporting the roles of these genes in lignin polymerization. In addition, none of the double mutants exhibited severe growth defects, such as shorter plant stature, dwarfing, or sterility, and their stems had improved cell wall degradability. This study will contribute to progress in lignin bioengineering to improve lignocellulosic biomass.