White-rot Fungus Pycnoporus Sanguineus Research Articles

Process intensification of cellulase and bioethanol production from sugarcane bagasse via an integrated saccharification and fermentation process

The production of value-added products such as cellulase and ethanol via a consolidated bioprocess could be realized by tapping into the multiple actions of a microbial community. For this purpose, an in situ saccharification and fermentation process through a sequential co-culture white-rot fungus and Saccharomyces cerevisiae on NaOH-pretreated sugarcane bagasse (SCB) was investigated. In the present work, white rot fungus plays a role in the production of cellulase enzymes. With the produced cellulase, an in situ saccharification process took place in the reactor to depolymerize pretreated SCB into reducing sugar. The reducing sugar was converted into ethanol via fermentation by S. cerevisiae, which was added into the system sequentially. White rot fungus Pycnoporus sanguineus was selected due to its competency in producing cellulase and reducing sugar production. The operating condition to maximize the production of reducing sugar in situ was obtained through a Central Composite Design method. A total of 3.13 g reducing sugar/100 g SCB was obtained when P. sanguineus was cultivated at 0.6% inoculum loading, 70% moisture content and 4 days. Subsequently, 4.5 g ethanol/100 g SCB was obtained from the in situ saccharification and fermentation system after S. cerevisiae was sequentially inoculated.

Deterioration of Wood Plastics Composites by the White-Rot Fungus Pycnoporus sanguineus

Wood plastic composites (WPC) are characterized by the mixing of wood fibers with plastics, allowing the production of new products whose characteristics are in several aspects superior to those of the original products and represent an expanding class of durable and low-cost materials in which their uses can reduce the environmental footprint and the dependence on petroleum products. Nevertheless, WPC has some setbacks, including biodegradation, which shortens its life span. In this study, the wood composite was exposed to the white-rot fungus Pycnoporus sanguineus in order to evaluate its resistance to biodegradation. The WPC was prepared with a 1:1 ratio of Eucalyptus spp. bark as reinforcement agent and polypropylene as matrix. Mechanical and rheological properties and mass loss were evaluated from 15 to 120 days of fungus exposure. After 15 days, a mass loss was detected, which transmitted a negligible effect on the impact resistance of the composite. For the 120-day fungus-exposed composite, the fungus produced a biofilm under the WPC that create a special environment for lignocellulosic consuming led to deterioration of the mechanical properties and minor changes on the thermal–chemical stability of the WPC. Finally, the study gave a great indication of the susceptibility of a Eucalyptus-based composite to biodegradation.

Rational design for fungal laccase production in the model host Aspergillus nidulans.

Laccases, multicopper oxidoreductases, are mainly produced in white-rot fungi and are considered as ideal green catalysts in industrial and biotechnological applications. However, the development of laccases is limited due to the slow growth of natural laccase producing strains and the low expression levels of laccases. In this study, we designed three regulation strategies for laccase gene expression in the model fungus Aspergillus nidulans. By introducing various promoters in front of the laccase gene pslcc from the white-rot fungus Pycnoporus sanguineus, we found that the laccase gene with the original promoter had effective expression in A. nidulans. Using the previously identified transcription factor RsmA regulatory mechanism, the aflR promoter was inserted into the pslcc expression vectors, and the laccase production was 15-fold higher in the strain overexpressing of RsmA compared to the control strain. To improve the laccase yield, the dipeptidyl-peptidase DppV, aspartic protease PepA and mannosyltransferase Mnn9 were successfully deleted in the A. nidulans host. The laccase activities were increased approximately 8-fold and 13-fold in the double deletions strains of Δmnn9ΔpepA and ΔdppVΔpepA over the control strains, respectively. Taken together, these results not only demonstrate an efficient system for heterologous protein production in the model fungus A. nidulans but also provide a general approach to applying regulatory methods to control gene expression.

Structural studies of two thermostable laccases from the white-rot fungus Pycnoporus sanguineus

Laccases are enzymes that have the ability to catalyze the oxidation of a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water. The active site of those proteins contains four copper ions, classified into three types. Laccases are interesting enzymes for study from the point of view of their structure, function and application because of their role in lignin degradation. Structural studies of two thermostable laccases produced by the strain Pycnoporus sanguineus CS43 (PsLacI and PsLacII) were performed. Both isoforms of PsLac show high thermal stability, at 60°C and 50°C, respectively, and they remained active at a high concentration of organic solvents. However, PsLacI has a higher thermal and pH stability and tolerance against inhibitors, and is a more efficient catalyst for ABTS and DMP (laccases substrate) than PsLacII. Based on the determined crystal structures we achieved insights into the structural factors relevant for the enzymatic properties of PsLacI and PsLacII. N-glycosylation site Asn354, which is very often present in structures of fungal laccases from other species, was not present in PsLac. This observation may be of particular significance due to the close distance between Asn354 and the substrate-binding pocket. This results in better access to the hydrophobic cavity for a particular substrate. Furthermore, we identified significant differences in the region of substrate-binding pocket, which confer PsLacI a markedly better performance than PsLacII.

Cytochemical and immunocytochemical characterization of wood decayed by the white rot fungus Pycnoporus sanguineus II. Degradation of lignin and non-cellulosic polysaccharides in European ash wood

Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) by the white rot fungus Pycnoporus sanguineus were investigated in European ash wood (hardwood) using transmission electron microscopy coupled with immunocytochemistry. Lignin was preferentially removed in secondary cell walls of ash xylem cells including fibers, vessel and (ray/axial) parenchyma cells prior to degradation of hemicelluloses (i.e. selective decay). Concomitantly, degradation of cell walls occurred by progressive thinning from the lumen surface outwards similar to simultaneous decay. Preferential degradation of middle lamellae before complete degradation of secondary cell walls (i.e. a typical feature of selective decay) was only observed in bi-/triseriate rays by initial attack from pit regions. Vessel and parenchyma cells showed greater resistance to decay than fibers with narrower decay zone produced. A comparison of decay between ash and spruce wood (Part I, softwood) demonstrated that P. sanguineus produces selective decay in both hard- and softwoods but more typical micromorphological features of selective decay are produced in spruce tracheids than ash fibers. This study highlights that degradation of lignin proceeds hemicelluloses in wood cell walls attacked by the selective white rot fungus P. sanguineus.

Cytochemical and immunocytochemical characterization of wood decayed by the white rot fungus Pycnoporus sanguineus I. preferential lignin degradation prior to hemicelluloses in Norway spruce wood

Degradation of lignin and non-cellulosic polysaccharides (pectins and hemicelluloses) in Norway spruce wood (softwood) by the white rot fungus Pycnoporus sanguineus was investigated using transmission electron microscopy coupled with immunocytochemistry. P. sanguineus produced selective decay in xylem cells including tracheids, ray tracheids and ray parenchyma cells. Lignin was preferentially removed first from cell walls with hemicelluloses (xylan and mannan) and cellulose remaining. Lignin in compound middle lamella (CML) regions of tracheids was also preferentially degraded prior to degradation of xyloglucan (hemicellulose). This differs from the previous concept of wood decay by selective white rot fungi demonstrating concomitant degradation of lignin and hemicelluloses. In contrast, no clear preferential degradation of lignin prior to pectins and hemicelluloses was observed in middle lamella cell corner (MLcc) regions of tracheids which were the last regions to be attacked by the fungus. Micromorphologically, prominent cell separation between tracheids by delignification of CML regions before complete degradation of secondary cell walls was observed as evidence of selective decay. Variations in resistance to decay by the fungus depending on cell wall orientation (radial vs. tangential) of tracheids and between cell types were also observed.

Improving the performance of a biofuel cell cathode with laccase-containing culture supernatant from Pycnoporus sanguineus

Laccases are multicopper oxidoreductases that can be used in biofuel cells to improve cathode performance by cathodic oxygen reduction. Here we present a laccase from the ligninolytic white-rot fungus Pycnoporus sanguineus that, in contrast to the Trametes versicolor laccase, can be produced in the absence of inducers in a standard culture medium. After 7days of cultivation the activity of this laccase in culture supernatant reached 2.5U/ml, which is high enough for direct application of the supernatant in biofuel cells. The highest current density of 115.0±3.5μA/cm2 at 400mV vs. SCE was obtained at pH 5 with a buckypaper cathode with a laccase-containing culture supernatant. The enzyme also showed electrocatalytic activity at pH 6 and 7. These results not only present a new cost-efficient laccase for improving cathode performance, but also show that new laccases with different catalytic properties can be suitable for biofuel cells.

Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design

The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate (CuSO4). The best conditions obtained from screening experiments in shaken flasks using tomato juice, CuSO4, and soybean oil were integrated in an experimental design. Enhanced levels of tomato juice as the medium, CuSO4 and soybean oil as inducers (36.8% (v/v), 3 mmol/L, and 1% (v/v), respectively) were determined for 10 L stirred tank bioreactor runs. This combination resulted in laccase titer of 143,000 IU/L (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), pH 3.0), which represents the highest activity so far reported for P. sanguineus in a 10-L fermentor. Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20,000 IU/L, whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times. Based on a partial characterization, the laccases of this strain are especially promising in terms of thermostability (half-life of 6.1 h at 60 °C) and activity titers.

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28°C, pH 4.5, an agitation speed of 600rpm and a dissolved oxygen concentration of ~25% of air saturation, the laccase productivity in the bioreactor exceeded 19UL(-1)days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325UL(-1).

Behavior of the Brown-rot Fungus Gloeophyllum trabeum on Thermally-modified Eucalyptus grandis Wood

In this study, we aimed evaluate the behavior of the brown-rot fungus Gloeophylum trabeum and white-rot fungus Pycnoporus sanguineus on thermally-modified Eucalyptus grandis wood. To this end, boards from five-year-eleven-month-old E. grandis trees, taken from the Duratex-SA company stock, were thermally-modified between 180 °C and 220 °C in the Laboratory of Wood Drying and Preservation at Universidade Estadual Paulista - UNESP, Botucatu, Sao Paulo state Brazil. Samples of each treatment were tested according to the ASTM D-2017 (2008) technical norm. The accelerated decay caused by the brown-rot fungus G. trabeum was compared with the decay caused by the white-rot fungus P. sanguineus, studied by Calonego et al. (2010). The results showed that (1) brown-rot fungus caused greater decay than white-rot fungus; and (2) the increase in temperature from 180 to 220 °C caused reductions between 28.2% and 70.0% in the weight loss of E. grandis samples incubated with G. trabeum.

Laccase production from oil palm industry solid waste: Statistical optimization of selected process parameters

AbstractOil palm frond parenchyma tissue was used as a solid substrate for the production of laccase via solid‐state fermentation using the white rot fungus Pycnoporus sanguineus. With a rectangular aluminium tray as solid‐state fermentation bioreactor, process parameters such as bed height, moisture and supplemented nitrogen (as urea solution) levels were studied and optimized using a statistical design of experiment. The moisture level exerted a significant effect on the process. The interaction effect observed between bed height and supplemented nitrogen level suggested that uniform distribution of supplemented nitrogen into the substrate bed was important. The proposed regression model sufficiently predicted the process response over the experimental range tested. The optimum parameter combination for laccase production was a 3‐cm bed height, 72% w/w moisture and 0.21% w/v supplemented nitrogen. Laccase productivity remained constant when the tray size was increased from 1.4 to 3.4‐fold.

Comparison of the characteristics of fungal and plant tyrosinases

Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, alpha-casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic L-tyrosine and diphenolic L-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein alpha-casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink alpha-casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink alpha-casein. In the reaction conditions studied, TrT showed the best ability to crosslink alpha-casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.

Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65 degrees C, respectively. The enzyme was stable up to 40 degrees C, and high laccase activity was maintained at pH 2.0-5.0. Sodium azide, L-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.

Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l(-1) for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l(-1).

Optimization of Laccase Production by Pycnoporus sanguineus in Submerged Liquid Culture

The white-rot fungus Pycnoporus sanguineus produces laccase under a range of C/N ratios in submerged liquid culture. Enzyme production was increased 50 fold in the presence of 20 μM xylidine to a m...

Optimization of laccase production by Pycnoporus sanguineus in submerged liquid culture

The white-rot fungus Pycnoporus sanguineus produces laccase under a range of C/N ratios in submerged liquid culture. Enzyme production was increased 50 fold in the presence of 20 μM xylidine to a maximum of 1368 U L-1 in a high carbon low nitrogen medium. Slight repression of enzyme production was observed in high nitrogen culture medium. Other potential inducers were less effective (Tween 80, wood fibres) and reduced the stimulation observed by xylidine alone when included in the same culture medium. Veratryl alcohol failed to stimulate laccase production. Activity of the enzyme activity in crude culture filtrate was stable at temperatures of 35 C and below with a pH optimum of 3.0. The laccase of P. sanguineus was identified as a ca 65 kDa protein produced as multiple isoforms.