Lignin degradation by peroxidase in organic media: A reassessment

Abstract

The effect of horseradish peroxidase/H202 in organic medium (dioxane/aqueous buffer, pH 5, 95:5) on the depolymerization of synthetic (dehydrogenatively polymerized) lignin was reinvestigated. In contrast to previous claims [Dordick, J. S., Marietta, M. A. & Klibanov, A. M. (1986) Proc. Natl. Acad. Sci. USA 83, 6255-6257], our results demonstrated that vigorous depolymerization of this substrate did not occur. Further, during this treatment ferulic acid was not a significant biodegradation product.It is well-established that certain fungi are capable of biodegrading structural polymers in plant material. Of particular interest are the white-rot fungi, such as Phanerochaete chrysosporium (1, 2), which can, to some extent, degrade lignin. Much effort has been expended towards understanding these biochemical conversions at the molecular level (3). In 1983, an enzyme (ligninase) isolated from P. chrysosporium was described as having powerful lignin-degrading capabilities (4). The substrate used for biodegradation in this study was not lignin per se, but instead an aqueous acetone extract of spruce wood, which had been methylated with 14CH31. More detailed investigations revealed that these ligninases were, in fact, peroxidase isoenzymes and, as such, did not vigorously degrade lignin. Indeed, it has now been reported that ligninase treatment of nonmethylated lignin results in both polymerization and depolymerization of the phenolic material (5). This is in general agreement with previous studies using horseradish peroxidase in aqueous media, from which it was concluded that although internal rearrangement of the lignin substrate can occur, the net effect was not that of lignin depolymerization (6). Thus, the report (7) that horseradish peroxidase/H202 in organic medium (dioxane/10 mM acetate buffer, pH 5, 95:5) could vigorously depolymerize both synthetic and natural lignin was so surprising as to warrant independent verification. MATERIALS AND METHODS Gel-Filtration Chromatography. A low-pressure liquid chromatography system was employed that used Sephadex LH-20 packed in an SR 10/50 column (Pharmacia). The Sephadex gel was swollen in the liquid phase for 3 hr before packing and then was equilibrated with six column volumes. Each column was calibrated with polystyrenes of molecular weight 2000, 800 (Pressure Chemical, Pittsburgh), and 1000 (Polymer Laboratory, Church Stretton, England) and dibenzyl malonate (molecular weight 284; Aldrich). The eluant was monitored at 280 nm, using an LKB Bromma 2158 Uvicord SD detector. Elution was either with eluant A (0.1 M LiCl in redistilled N,N-dimethylformamide) or eluant B (anhydrous dioxane).Purification of Dioxane. A mixture of dioxane (3 liters) plus concentrated HCl (37.5 ml) in water (300 ml) was refluxed for 12-18 hr under N2. The solution was cooled to room temperature and excess solid KOH was added with stirring. The dioxane was decanted from the thick, dark aqueous layer that resulted and was stored over KOH (150 g of KOH per liter of dioxane). The dioxane...

Chemical assesment of lignin biodegradation some qualitative and quantitative aspects

“…Using GC-MS according to Lapierre [31], Table 4 also shows a preferential relative decrease in /3-5 and /3-1 dimeric units as compared to the relative changes in 5-5' and 4-0-5' units, suggesting a higher susceptibility of these dimeric structure to enzymatic oxidation, the mechanism of which remains unknown. Whatever the differences and even when gel permeation experiments did not allow to demonstrate any biomimetic degradation by HRP, in agreement with Lewis et al [26], Table 4 allows to conclude that HRP also exhibits ligninolytic properties, in agreement with Dordick et al [27].…”

“…For example, a plant peroxidase from horseradish has been reported to vigorously depolymerize milled wood lignin in organic solvent containing few percent of aqueous buffer while being unable, however, to degrade lignins in aqueous solution [25]. Later on, this statement however has been reassessed by Lewis et al [26] who, in contrast to these previous claims, reported that vigourous depolymerization of synthetic lignin (DHP: dehydrogenatively polymerized) did not occur in aqueous organic media.…”

Biodegradation

“…The breakdown of lignin by peroxidase in non-polar solvents forming quantities of compounds such as ferotic acid and vanillin was described by . However, Lewis et al (1987) could not repeat these results and there have been no further claims in this area to date.…”

Biotechnology in the degradation and utilization of lignocellulose

“…It has been suggested that peroxidases can depolymerize lignin more effectively in non-aqueous solvents (Dordick et al 1986). If this is indeed the case (Lewis et al 1987), it has relevance to the technological exploitation of lignin peroxidases and lignins. 7.…”