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To efficiently decompose biomass, fungi have developed various enzymatic and non-enzymatic strategies and are a source of versatile biocatalysts. The endoglucanases in glycosyl hydrolase CAZy family 45 (GH45) are known for their small size, a high thermostability and a broad substrate specificity that has been employed in textile and detergent industries. Here we report the heterologous expression and characterisation of an GH45 endoglucanase from the brown rot Fomitopsis pinicola and its direct comparison to an already characterised GH45 from the white rot Phanerochaete chrysosporium. Both enzymes were recombinantly expressed in Pichia pastoris and purified by two chromatographic steps. The biochemical characterisation highlighted the acidophilic character, with an optimal pH of 4, and a preference for amorphous substrates as carboxymethyl cellulose (CMC) and substrates containing β-1,4-glucans rather than the previously reported β-1,3/1,4-glucans lichenan and β-glucan. The dominating products from β-1,4-glucans were C3-C6 oligosaccharides, whereas from β-1,3/1,4-glucans glucose was the main reaction product. From the characterisation no differences between the brown rot and the white rot GH45 was evident.

Laccase is predominantly found in lignin degrading filamentous white rot fungi, where it is involved in the oxidative degradation of this recalcitrant heteropolymer. In brown rot fungi it is much less prevalent: laccases from only a few brown rots have been detected and only two have been characterized. This study tries to understand the role of this ligninolytic enzyme in brown rots by investigating the catalytic properties of laccases secreted by Fomitopsis pinicola FP58527 SS1. When grown on either poplar or spruce wood blocks, several laccases were detected in the secretome. Two of them (FpLcc1 and FpLcc2) were heterologously produced using Trichoderma reesei QM9414 Δxyr1 as expression host and purified to homogeneity by consecutive steps of hydrophobic interaction, anion exchange and size exclusion chromatography. With the substrates 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol both laccases showed similar, low pH-optima below 3 for ABTS and 2,6-DMP and at pH 3.5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined KM values were low while kcat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid.

Versatile peroxidase (VP) from Bjerkandera adusta is an enzyme able to oxidize bulky and high-redox substrates trough a Long-Range Electron Transfer (LRET) pathway. In this study, the introduction of radical-forming aromatic amino acids by chemical modification of the protein surface was performed, and the catalytic implications of these additional surface active-sites on the oxidation of 2,6-dimethylphenol, Mn2+ and Remazol Brilliant Blue R (RBBR) were determined. These three different substrates are oxidized in different active-sites of enzyme molecule, of which the high redox RBBR the only one that is transformed by an external radical formed on the protein surface. Both catalytic constants kcat and KM were significantly affected by the chemical modifications. Tryptophan- and tyrosine-modified VP showed higher catalytic transformation than the unmodified enzyme for RBBR, while the Mn2+ oxidation was significantly reduced by all chemical modifications. Electron Paramagnetic Resonance studies demonstrated the formation of additional protein-based radicals after the chemical modification with radical-forming amino acids. In addition, the catalytic rate of the LRET-mediated transformation showed a good correlation with the ionization energy of the additional amino acid on the protein surface.

The inhibitor and substrate specificities of versatile peroxidase from Bjerkandera fumosa (VPBF) were studied. Two different effects were found: NaN(3), Tween-80, anthracene, and fluorene decreased the activity of VPBF, but p-aminobenzoic acid increased it. A mixed mechanism of effector influence on the activity of this enzyme was shown. The catalytic properties of VPBF in the oxidation of mono- and polycyclic aromatic compounds were studied also. 2,7-Diaminofluorene, ABTS, veratryl alcohol, and syringaldazine can be oxidized by VPBF in two ways: either directly by the enzyme or by diffusible chelated Mn(3+) as an oxidizing agent. During VPBF oxidation of 2,7-diaminofluorene, both with and without Mn(2+), biphasic kinetics with apparent saturation in both micromolar and millimolar ranges were obtained. In the case of ABTS, inhibition of VPBF activity by an excess of substrate was observed. Direct oxidation of p-aminobenzoic acid by versatile peroxidase was found for the first time. The oxidation of three- and four-ring PAHs by VPBF was investigated, and the oxidation of anthracene, phenanthrene, fluorene, pyrene, chrysene, and fluoranthene was shown. The products of PAH oxidation (9,10-anthraquinone, 9,10-phenanthrenequinone, and 9-fluorenone) catalyzed by VPBF were identified.

The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield. Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes. Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5′-adenosyl)- l -methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones.

The effects of biological pretreatment on Pinus radiata and Eucalyptus globulus, were evaluated after exposure to two brown rot fungi Gloephylum trabeum and Laetoporeus sulphureus. Changes in chemical composition, structural modification, and susceptibility to enzymatic hydrolysis in the degraded wood were analyzed. After eight weeks of biodegradation, the greatest loss of weight and hemicellulose were 13% and 31%, respectively, for P. radiata with G. trabeum. The content of glucan decreased slightly, being the highest loss of 20% for E. globulus with G. trabeum. Consistent with degradation mechanism of these fungi, lignin was essentially undegraded by both brown rot fungi. Both brown rot fungi cause a sharp reduction in the cellulose degree of polymerization (DP) in the range between 58% and 79%. G. trabeum depolymerized cellulose in both wood faster than L. sulphureus. Also, structural characteristic of crystalline cellulose were measured by using two different techniques - X-ray diffraction (XRD) and infrared spectroscopy (FT-IR). The biological pretreatments showed an effect on cellulose crystallinity structure, a decrease between 6% and 21% was obtained in the crystallinity index (CrI) calculated by IR, no changes were observed in the XRD. Material digestibility was evaluated by enzymatic hydrolysis, the conversion of cellulose to glucose increased with the biotreatment time. The highest enzymatic hydrolysis yields were obtained when saccharification was performed on wood biopretreated with G. trabeum (14% P. radiata and 13% E. globulus). Decreasing in DP and CrI, and hemicellulose removal result in an increase of enzymatic hydrolysis performance. Digestibility was better related to DP than with other properties. G. trabeum can be considered as a potential fungus for biological pretreatment, since it provides an effective process in breaking the wood structure, making it potentially useful in the development of combined pretreatments (biological-chemical). A viable alternative to pretreatment process that can be used is a bio-mimetic system, similar to low-molecular complexes generated by fungi such as G. trabeum combined pretreatments (biological-chemical).

Two acidic β-glucosidases (βGI and βGII) from the brown rot fungus Fomitopsis palustris were purified to homogeneity by several chromatographic steps. βGI and βGII had molecular weights of 130 and 213 kDa, respectively, and exhibited optimum activity at pH 2.5 and 55°C. The K(m) values of βGI and βGII for p-nitrophenyl-β-d-glucopyranoside were 0.706 and 0.971 mM, respectively. Although the effect of metal ions and inhibitors differed between the two enzymes, both β-glucosidases exhibited preferential glucose release during hydrolysis of cello-oligosaccharides, indicating that βGI and βGII possess effective exo-type activities. Notably, F. palustris was able to produce ethanol when cultured on medium containing 20 g/l of glucose, mannose, cellobiose, and maltose, in which the maximum ethanol concentrations measured were 9.2, 8.7, 9.0, and 8.9 g/l, corresponding to 90.2%, 85.3%, 88.2%, and 87.3% of the theoretical yield, respectively. These findings suggest that F. palustris has the ability not only to secrete β-glucosidase enzymes effective at low pH, but also to function as a biocatalyst, which may be suitable for the conversion of lignocellulosic materials into ethanol.