Terhi K. Hakala

Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus

The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements. Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures of P. rivulosus. Both laccases showed thermal stability at up to 60°C. Lac-4.8 was thermally activated at 50°C. Surprisingly, both laccases displayed atypically low pH optima (pH 3.0–3.5) in oxidation of the commonly used laccase substrates syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine), 2,6-dimethoxyphenol and guaiacol (2-methoxyphenol). Steady-state kinetic measurements pointed to unusually low affinity to guaiacol at low pH, whereas the kinetic constants for the methoxyphenols and ABTS were within the ranges reported for other fungal laccases. The combination of thermotolerance with low pH optima for methoxylated phenol substrates suggests that the two P. rivulosus T241i laccases possess potential for use in biotechnological applications.

Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus

Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn2+, veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn2+ and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.

Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata

Lignin peroxidase (LiP) is the first enzyme connected to oxidative breakdown of the aromatic plant heteropolymer lignin and related xenobiotics. However, this extracellular enzyme has been described in only a few species of wood-decaying basidiomycetous fungi. The white rot basidiomycete Phlebia radiata 79 readily produces a versatile set of lignin-oxidizing enzymes including lignin and manganese peroxidases (LiPs and MnPs) and laccases. Here we describe genomic and primary structure of two new LiP-encoding genes, Pr-lip1 and Pr-lip4, and genomic characterization for isozyme LiP3/LIII of P. radiata, encoded by the gene depicted Pr-lip3. Pr-lip1 and Pr-lip4 code for 370- and 361-amino-acid long proteins beginning with 26- and 24-amino-acid secretion pre-propeptides, respectively. Translated LiP1 and LiP4 share the highest protein sequence identity (74 and 86%) with P. radiata LiP3, and 70% identity with the one deduced LiP from Bjerkandera adusta. The three P. radiata LiP sequences form a coherent phylogenetic cluster, which is further supported by similarities within gene organization interrupted by 11-introns. To find out the significance of LiP upon fungal growth on natural lignocellulose, such as wood, we studied ligninolytic gene expression on hardwood (milled alder) and softwood (spruce chips). All the LiP-encoding genes were expressed on wood with predominance of Pr-lip3 transcript abundance, in particular on spruce wood chips, where also time-dependent expression of the multiple lip genes was observed.