Sari Galkin

Production of organic acids and oxalate decarboxylase in lignin-degrading white rot fungi

Intracellular oxalate decarboxylase (ODC, EC 4.1.1.2) activity was screened in the mycelium of 12 white rot fungi. ODC activity was detected in the mycelial extracts of Dichomitus squalens, Phanerochaete sanguinea, Trametes ochracea, and Trametes versicolor (strain R/7) after addition of 5-mM oxalic acid to the liquid culture medium. In D. squalens, intracellular ODC activity increased six-fold with addition of oxalic acid. Production of extracellular organic acids by the four ODC-positive fungi was followed in liquid cultures and in solid state cultures of spruce wood chips by using HPLC and capillary zone electrophoresis (CZE). The four ODC-positive fungi secreted oxalic acid both in liquid and solid state cultures showing different production patterns until the end of growth (31 days). Upon cultivation on solid spruce wood chips, manganese peroxidase (MnP) activity peaked simultaneously in these fungi with the accumulation of extracellular oxalic acid. In addition to oxalic acid, glyoxylic and formic acids were detected in the cultures of D. squalens.

Production of organic acids by different white-rot fungi as detected using capillary zone electrophoresis

Capillary zone electrophoresis was used as an analytical method for the first time to determine organic acids accumulated by 15 white-rot fungi both in liquid and solid lignocellulose medium. Oxalic acid accumulation by Phanerochaete chrysosporium F1767 (ATCC 24725), Phlebia radiata 79 (ATCC 64658) and Ceriporiopsis sub-vermispora FP-90031-sp in the solid medium was maximal during the first and second weeks of growth.

Mineralization and solubilization of synthetic lignin by manganese peroxidases from Nematoloma frowardii and Phlebia radiata

Crude and purified manganese peroxidase from the white-rot fungi Nematoloma frowardii and Phlebia radiata catalyzed the partial depolymerization of a [14C-ring]labelled synthetic lignin into water-soluble fragments (30–50%). The in vitro depolymerization of the 14C-labelled lignin was accompanied by a release of 14CO2 ranging from 4 to 6%. Small quantities of the thiol mediator glutathione stimulated the depolymerization of lignin resulting in a mineralization and solubilization of up to 10 and 64%, respectively. Most of the water-soluble substances formed had molecular masses around 0.7 kDa, although a higher-molecular mass fraction was also detectable (>2 kDa). Photometric assays using 2,2′-azinobis(3-ethylbenzothiazolinesulphonate) as an indicator demonstrated that high levels of Mn(III), which were very probably responsible for the depolymerization and mineralization of the 14C-labelled lignin, were adjusted within the first 24 h of incubation. The manganese peroxidase catalyzed depolymerization process was not necessarily dependent on H2O2; also in the absence of the H2O2-generating system glucose/glucose oxidase, effective solubilization and mineralization of lignin dehydrogenation polymerizate occurred, due to the in part superoxide dismutase sensitive, ‘oxidase-like’ activity of MnP which probably produces radical species and peroxides from malonate.

The laccase-catalyzed modification of lignin for enzymatic hydrolysis.

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.

Characterisation of milled wood lignin from reed canary grass (Phalaris arundinacea)

Lignin samples from reed canary grass (Phalaris arundinacea) were prepared by milling in the dry state (MWL) or in toluene (TMWL). Further, enzymatically liberated lignin (EL) was prepared from the dioxane-water insoluble residue from the preparation of MWL. The NMR spectra of the lignin preparations revealed the presence of both guaiacyl and syringyl units, as well as p-hydroxyphenyl structures. The most prominent side chain linkages were identified using HMQC. In the 13 C NMR and FTIR spectra signals typical for p-coumaric esters could be observed. Permanganate oxidation revealed that some of the p-hydroxyphenyl structures were incorporated in the lignin, together with guaiacyl and syringyl units. The lignin preparations had very similar molecular weight distributions and structural composition. These lignin preparations are distinguished by high amounts of β-O-4 structures and by low amounts of phenolic hydroxyls, resinol units (β-β) and of condensed units (β-5 and 5-5). This trait was even more pronounced in an ether soluble fraction of the MWL that contained almost exclusively side chain linkages of the β-O-4 type. This difference in distribution of structural units indicates some structural heterogeneity in reed canary grass lignin.

Effect of phenolic acids on manganese peroxidase-dependent peroxidation of linoleic acid and degradation of a non-phenolic lignin model compound

The influence of aromatic phenolic and non-phenolic acids on manganese peroxidase (MnP)-dependent peroxidation of linoleic acid, and oxidation of a non-phenolic lignin model compound (LMC) was studied. Phenolic compounds inhibited both the MnP-dependent lipid peroxidation (LPO) and non-phenolic LMC degradation in the system. The antioxidant activity of the aromatic compounds in the enzymatic system with MnP-dependent LPO depends on the presence of the phenolic hydroxyl groups attached to the aromatic ring structure, the methoxylation of the hydroxyl group in the ortho position in diphenolics, and number of carbon atoms in the side chain. Natural phenolic compounds inhibit the oxidation of non-phenolic lignin in the system based on MnP-mediated LPO, but do not prevent it. This result indicates that MnP-mediated LPO may play an important role in lignin degradation even in the presence of the phenolic antioxidant compounds, and supports the possibility of the involvement of LPO in the degradation of lignin in wood.

Enzymatic Modification of Flaxseed Fibers

Flaxseed (Linum usitatissimum L.) fibers were modified by oxidoreductive and cellulolytic enzymes. The lignin amount and intrinsic plant peroxidase activity was evaluated by histochemical and spectrophotometric assays. Peroxidase activity was not found from bast fibers. The flaxseed fibers were further separated and treated with laccase to conjugate the model compounds, that is, the hydrophobic gallate molecules on fiber surfaces. Laccase was able to slowly oxidize fiber-conjugated phenolics, but no fundamental changes in fiber cell surface structure or notable coupling of the applied hydrophobic gallate molecules onto the fibers occurred, as revealed by Fourier transform infrared spectroscopy. The reactivity of the mature fibers was further investigated using cellulolytic enzymes. Cellobiohydrolase (CBH) and endoglucanase (EG)-rich enzyme preparations were applied to reach a hydrolysis degree of 1-6% (of the fiber dry matter) using a standard enzyme dosage. The CBH mixture altered the fiber surface morphology distinctly, and SEM images illustrated fibers in which the cellulose fibrils seemed to be loosened and partially hydrolyzed. In contrast, the effect of the EG-rich preparation without CBH activity was notable on the fiber surface, polishing the surfaces. The cellulolytic treatments were potentially interesting for specific enzymatic modifications of flax fiber surfaces, whereas the approach to use oxidoreductive enzyme treatments on mature linseed fibers offered little potential, obviously due to the low lignin content of the fibers.