0000000000637589
AUTHOR
Johanna Buchert
Activity of laccase on unbleached and bleached thermomechanical pulp
The introduction of value-added properties to pulp fibres is an attractive proposition. One interesting option is targeted modification of fibre surfaces by enzymatic activation. In this work, the activity of laccase on pulp and pulp fractions from thermomechanical pulp (TMP) and peroxide bleached TMP was studied on the basis of consumption of the co-substrate oxygen in the reaction and by studying the formation of radicals in the pulp material as analysed by electron paramagnetic resonance spectroscopy (EPR). Laccases obtained from Trametes hirsuta and Myceliophthora thermophila were used in the study. Laccases were found to be active on pulp material of unbleached TMP, whereas only fines …
Characterization of sulfhydryl oxidase from Aspergillus tubingensis
Background Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear. Results The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initia…
Additional file 7: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis
Part of the alignment of 25 sequences from the same protein family (InterPro IPR000103). On the first line is shown AtSOX retrieved from A. tubingensis genome. The C-X-X-C motifs are marked with a box. The sequences: AtSOX, secreted SOX from A. tubingensis; AnSOX, secreted SOX from A. niger; AoSOX, secreted SOX from A. oryzae; DepH, enzyme from C. violaceum; GliT, enzyme from A. fumigatus; HlmI, enzyme from S. clavuligerus. The other abbreviations are shown in the legend of Fig. 7. (PPTX 95Â kb)
Additional file 3: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis
AtSOX activity measured by HVA-peroxidase coupled assay using reduced GSH (5 mM) as a substrate according to [33]. The enzyme reaction is at the enzymatic rate (linear area ca. 0 - 150 s). The production of the fluorescent HVA dimer was followed at excitation wavelength 320 nm and emission wavelength 420 nm. The reduced AtSOX activity with the inhibitor zinc sulphate (10 mM) is also shown (dashed line). The triplicate measurements were done. (PPTX 121 kb)
Additional file 2: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis
AtSOX activity measured by oxygen consumption assay using 3 mM reduced GSH as a substrate (continuous line). The reaction occurred at the enzymatic rate (the linear area ca. 0.5 - 3.5 min). The amount of dissolved oxygen in the reduced GSH solution prior addition of enzyme is shown with a dashed line. The triplicate measurements were done. (PPTX 6691 kb)
Additional file 4: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis
Absorbance spectra (ca. 10 min) of 5 mM reduced GSH (a.) and 5 mM reduced GSH with AtSOX (b.). Arrow indicates the direction of increased UV adsorption due to enzymatic oxidation of the substrate. (PPTX 395 kb)
Additional file 1: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis
Purification of AtSOX as analysed by SDS-PAGE. Molecular weight (MW) standards are shown in lanes 1 and 9. The sample from initial crude cell-free medium is shown in lane 2. As a first purification step was used anion exchange chromatography with a Q Sepharose column. In lane 3 are the unbound proteins, and in the lanes 4â 6 bound and then eluted proteins, from Q Sepharose column. Lane 6: AtSOX containing fractions selected for further purifications steps. In the lanes marked 7 and 8 are shown fractions obtained from the last purification step using anion exchange chromatography with Resource Q column (analysed in a separate SDS-PAGE gel with MW standards in lane 9). (PPTX 137Â kb)
Additional file 6: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis
For each chromosomal cluster the table shows accession numbers for genes (Accession), scaffold identifier (Scaffold), start and end on the scaffold, direction of the gene (Direction) and Interpro protein domain identifiers found in the genes (Interpro), as details for Additional file 5. (XLSX 20Â kb)
Additional file 5: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis
Details to Fig. 6 Candidate secondary metabolism clusters with SOX enzymes on fungal chromosomes. On the left an approximate phylogenetic tree of the species compiled from literature [54, 55]. On the right a stretch of a scaffold from each species containing the cluster and neighbouring genes. Genes are shown as boxes on the scaffold stretch. NRPS, PKS, P450 and Zn2 are indicated when present. Grey lines connect genes with identical protein domains on adjacent scaffolds (excluding NRPS, PKS, P450, Zn2 and SOX genes) in order to reveal syntenies. Codes above the gene boxes are their identifiers and below them the Interpro protein domain identifiers found in the genes. Panel a. shows the glio…