Chemical modifications of laccase from white-rot basidiomycete Cerrena unicolor.

Laccases belong to the group of phenol oxidizes and constitute one of the most promising classes of enzymes for future use in various fields. For industrial and biotechnological purposes, laccases were among the first enzymes providing larger-scale applications such as removal of polyphenols or conversion of toxic compounds. The wood-degrading basidiomycete Cerrena unicolor C-139, reported in this study, is one of the high-laccase producers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability or stability in broad ranges of pH. In this work, modifications of laccase isoforms by hydrophobization, hydrophilization, and polymerization were performed. The hydrophobized and hydrophilized enzyme showed enhanced surface activity and higher ranges of pH and temperatures in comparison to its native form. However, performed modifications did not appear to noticeably alter enzyme's native structure possibly due to the formation of coating by particles of saccharides around the molecule. Additionally, surface charge of modified laccase shifted towards the negative charge for the hydrophobized laccase forms. In all tested modifications, the size exclusion method led to average 80 % inhibition removal for hydrophilized samples after an hour of incubation with fluoride ions. Samples that were hydrophilized with lactose and cellobiose showed an additional 90 % reversibility of inhibition by fluoride ions after an hour of concluding the reaction and 40 % after 24 h. The hydrophobized laccase showed higher level of the reversibility after 1 h (above 80 %) and 24 h (above 70 %) incubation with fluoride ions. The addition of ascorbate to laccase solution before a fluoride spike resulted in more efficient reversibility of fluoride inhibitory effect in comparison to the treatments with reagents used in the reversed sequence.

Influence of aromatic compounds on biodegradation of [14C]-labeled xylan and mannan by the white-rot fungus Phlebia radiata.

Radiolabeled [14C]arabinoxylan from wheat meal and [14C]galactoglucomannan from red clover meal were prepared by using 14CO2 as a precursor. Twice as much mannan was mineralized than xylan after 14 days of incubation with Phlebia radiata. Low-molecular-weight phenolic compounds structurally related to lignin increased during mineralization of both hemicellulose fractions. Veratryl alcohol increased degradation of arabinoxylan by approximately 28.5%, whereas veratric acid increased it by only 9.0%. Vanillic acid and ferulic acid also stimulated degradation by 16.6% and 34.7%, respectively. Veratryl alcohol and ferulic acid increased degradation of galactoglucomannan by approximately 75%. Veratraldehyde in both cases repressed the degradation process (23.6% arabinoxylan, 43.8% galactoglucomannan). These results indicate that the degradation of hemicelluloses, e.g., xylan and mannan, by P. radiata is enhanced by addition of aromatic compounds.

Demethylation of [14C]-labelled veratric acid and oxidation of methanol and formaldehyde by the white-rot fungus Phlebia radiata.

Veratric acids 14C-labelled in carboxyl group, 3-OCH3, 4-OCH3, or aromatic ring together with unlabelled veratric acid were supplemented in the cultures of the white-rot fungus Phlebia radiata. The effect of various carbon sources on the release of 14CO2 was studied. Veratric acid was readily decarboxylated, maximally already on day 1 from the addition of [14COOH]-veratric acid. High amounts (4%) of glucose slightly repressed the decarboxylation. In medium supplemented with cellulose the methoxyl group in position 4 was much more readily mineralized to CO2 than the group in position 3. The maximum evolution was achieved on day 5, two days from the addition. Cellulose did not repress methanol oxidation but repression of methanol oxidation by glucose was detected in media supplemented with [O14CH3]-veratric acids and 14CH3OH. However, glucose did not repress oxidation of H14CHO. The apparent uptake of 14C by fungal mycelium, especially from methoxyl groups, but also from the aromatic ring, may partially be due to the strong slime formation observed in cellobiose medium. Also in cellobiose medium apparent uptake of 14C from 14C-labelled methoxyl groups was observed.

Isolation and properties of elongation factor 1 from Saccharomyces cerevisiae.

Polypeptide elongation factor 1 was isolated from yeast postribosomal supernatant. The highly purified factor was resolved on Ultrogel AcA-44 into two complementary fractions. One of these fractions contained two different polypeptide chains corresponding to a Ts-like elongation factor EF-1 beta gamma. The other fraction represented the light form of the factor, designated EF-1 alpha, with a molecular weight of approximately 50,000. The obtained results indicate that EF-1 from lower eukaryotes is also composed of three distinct polypeptides.