Pekka Maijala

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.

Enzymatic accessibility of fiber hemp is enhanced by enzymatic or chemical removal of pectin.

Pectinolytic enzymes, steam explosion and alkaline treatment were used to assess the role of pectin for the accessibility of hydrolytic enzymes in the enzymatic hydrolysis of biomass. Hemp (Cannabis sativa L.), a potential energy crop especially in boreal climate with a low need of fertilizers, was used in the study either as untreated or anaerobically preserved raw material. Addition of pectinases increased the hydrolysis yield by 26%, 54%, and 64% from the theoretical carbohydrates of untreated, acid, and alkali-preserved materials, respectively. Steam explosion and hot alkali treatment increased the conversion of the total carbohydrates by 78% and 60%, respectively, compared to the untreated hemp. Elevated separation of cells within the hemp stalk tissues and an increased surface area was revealed after hot alkali or pectinase treatments, contributing to the increased conversion to sugars by commercial enzymes.

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.

Characterization of hemicellulases from thermophilic fungi

The thermophilic fungi Thermomyces lanuginosus, Malbranchea cinnamomea, Myceliophthora fergusii and the thermotolerant Aspergillus terreus were cultivated on various carbon sources, and hemicellulolytic and cellulolytic enzyme profiles were evaluated. All fungi could grow on locust bean galactomannan (LBG), Solka floc, wheat bran and pectin, except T. lanuginosus, which failed to utilize LBG for growth. Different levels of cellulase and hemicellulase activities were produced by these fungal strains. Depending on the carbon source, variable ratios of thermostable hydrolytic enzymes were obtained, which may be useful in various applications. All strains were found to secrete xylanolytic and mannanolytic enzymes. Generally, LBG was the most efficient carbon source to induce mannanase activities, although T. lanuginosus was able to produce mannanase only on wheat bran as a carbon source. Xylanolytic activities were usually highest on wheat bran medium, but in contrast to other investigated fungi, xylanase production by M. fergusii was enhanced on pectin medium. Preliminary thermostability screening indicated that among the investigated species, thermotolerant glycosidases can be found. Some of the accessory activities, including the α-arabinosidase activity, were surprisingly high. The capability of the produced enzymes to improve the hydrolysis of lignocellulosic pretreated substrate was evaluated and revealed potential for these enzymes.

Acrebol, a novel toxic peptaibol produced by an Acremonium exuviarum indoor isolate

Aims:  To identify a toxin and its producer isolated from woody material in a building where the occupants experienced serious ill health symptoms.

Syringyl-type simple plant phenolics as mediating oxidants in laccase catalyzed degradation of lignocellulosic materials: Model compound studies 10th EWLP, Stockholm, Sweden, August 25–28, 2008

Abstract The utility of simple syringyl-type phenolics of plant origin and some common synthetic compounds were compared with regard to their ability to act as laccase mediators in the oxidation of non-phenolic lignin model compounds. It was demonstrated that syringyl-type phenols, especially acetosyringone and methyl syringate, were able to mediate the oxidation of substrates of high oxidation potential by a laccase with a low redox potential. A mediator dose around 10% showed good performance compared to the equimolar quantities needed when the synthetic compounds 1-hydroxybenzotriazole or N-hydroxyacetanilide were used.

Improved efficiency in screening for lignin-modifying peroxidases and laccases of basidiomycetes

Background: Wood rotting white-rot and litter-decomposing basidiomycetes form a huge reservoir of oxidative enzymes, needed for applications in the pulp and paper and textile industries and for bioreme-

Enzymatic pretreatment of seed flax- and polylactide-commingled nonwovens for composite processing

Commingled composites of polylactide (PLA) and seed flax fibers were prepared by carding and needle pressing of PLA and flax fibers to nonwoven mats followed by compression molding. The nonwoven mats were treated using two different types of cellulolytic enzymes, namely cellobiohydrolases and endoglucanases. Both the enzymes changed the properties of the seed flax fibers surface. The resulting composites were studied using scanning electron microscopy, mechanical testing and thermal analysis. The effects of these modifications were minor on the thermal and mechanical properties measured. However, in the scanning electron micrographs, a difference in the fiber pull-out behavior of the untreated and treated fibers was observed. Treated seed flax fibers showed an improvement in adhesion with PLA.