RNA-binding proteins (RBP) shuttle between cellular compartments either constitutively or in response to stress and regulate localization, translation and turn over of mRNAs. In our laboratory, cytosolic proteome map of Phanerochaete chrysosporium was established and upon Pb exposure, the changes in cytosolic protein expressions were determined. The identified RBPs were a newly induced polyadenylate-binding protein (RRM superfamily) as well as two up-regulated proteins, namely splicing factor RNPS1 and ATP-dependent RNA helicase, all being very important candidates of post-transcriptional control in response to stress. This finding inspired us to conduct Real Time PCR studies in order to have a better understanding of the changes in the expression of corresponding genes at mRNA level in response to Pb exposure, thus the present study aims at examining the effect of lead exposure on the transcript levels of the genes coding for ATP-dependent RNA helicase, splicing factor RNPS1 and polyadenylate binding protein. As shown via expression analysis based on Real Time PCR, the mRNA level of splicing factor RNPS1 showed 2.68, 2.62 and 4.86 fold increases in a dose-dependent manner when the cells were grown for 40 h in the presence of 25, 50 and 100 &micro / M Pb, repectively. ATP-dependent RNA helicase mRNA level showed no significant increase in response to 25 &micro / M Pb exposure while increased 2 and 1.84 fold in response to 50 and 100 &micro / M Pb, respectively. Polyadenylate binding protein mRNA levels revealed no significant increase when exposed to 25, 50 and 100 &micro / M Pb. As to the mRNA dynamics as a function of duration of lead exposure, the mRNA level of this protein showed 2.54-fold increase upon 1 h exposure to 100 &micro / M Pb. Splicing factor RNPS1 mRNA level showed a significant increase of 19.22 fold at 2nd h of 50 &micro / M Pb exposure. Expression level of ATP-dependent RNA helicase was not affected by the time of exposure to Pb.

Biosorption is a phenomenon involving the mechanisms that basically mediate heavy metal tolerance of microorganisms as well as sequestration of heavy metals from environment. Different classes of microorganisms have different biosorption capacities, as a result of the differences in composition and types of functional groups found on cell surfaces. The present study was undertaken to identify the molecular mechanisms for lead [Pb(II)] biosorption in the white-rot fungus, Phanerochaete chrysosporium. The methodology involved selective blocking of the functional groups known to participate in heavy metal biosorption and allowed us to determine their relative roles in Pb (II) biosorption in this organism. The relative concentrations of the Pb (II) sorbed from the aqueous environment and Mg2+ and Ca2+ ions released to the aqueous environment were measured and compared with both native and chemically-modified biomasses by using atomic absorption spectroscopy. Fourier-Transform Infrared (FTIR) spectroscopy technique was used to monitor and analyze the molecular-level changes in both native and chemically modified cell surfaces upon Pb (II) exposure. Interactions of Pb (II) with the biomass surface was determined by observing the changes in wavenumber and absorbance of NH stretching and Amide I bands arising from the amine groups and C=O stretching band arising from the carboxyl groups. The roles of phosphate groups and lipids were also investigated. Carboxyl groups seemed to be the most important functional groups for Pb (II) biosorption in P. chrysosporium, since the biosorption capacity dramatically decreased (by 92.8 %) in carboxyl groups-blocked biomass. Amine groups were found to play a secondary and minor role in Pb (II) biosorption, only a slight decrease (6 %) in Pb (II) biosorption was detected with amine groups-blocked biomass. Blocking of phosphate groups provided a small increase in biosorptive capacity and did not appear to have much significant role in biosorption. Upon chemical treatment with acetone to extract lipids of the cell surfaces, an increase of 20.3 % in the Pb (II) biosorptive capacity was determined. It was concluded that carbonyl and carboxyl groups of chitin and glucan are the major sites and ion exchange via these groups is the main mechanism for Pb (II) biosorption in P. chrysosporium.

Thesis (MTech (Chemical Engineering))--Cape Technikon, 2000. / The large quantities of wood chips produced at mines from damaged underground timber contain gold that cannot be completely recovered by cyanidation. A fungus that can degrade a portion of the wood matrix will allow the gold that was previously locked up, to come into contact with the cyanide solution during beneficiation, thereby improving recoveries. The fungus Phanerochaete chrysosporium produces enzymes that use the organic compounds found in lignin as substrate. Consequently, the fungus is able to selectively break down lignin, which is one of the major components of wood. Chips sampled from Vaal Reef Mine contained between 2 and 5 mg/kg gold. The main source of gold in the chips was determined to be impregnated gold-bearing ore and discrete gold particles. Direct cyanidation resulted in around 60 per cent recovery prior to biological treatment. Despite relatively high weight losses caused to the chips as a result of treatment with Phanerochaete chrysosporium gold recovery only increased 10 per cent after 4 weeks treatment compared to direct recovery without treatment.

Le sol est une interface complexe entre tous les compartiments de l'environnement. Leur pollution participe à la diffusion de nombreux polluants. Les polychlorobiphényles sont des molécules toxiques persistantes dans l'environnement. Largement utilisés notamment dans les huiles diélectriques, ils contaminent aujourd'hui de nombreux sols industriels. Le traitement thermique de ces sols est très onéreux et peut entraîner l'émission de dioxines. L'objectif de ce travail est d'étudier un procédé de traitement biologique pour la dégradation des PCB dans les sols. Une dégradation biologique de PCB a été observée en présence de deux organismes cultivés, Burkholderia xenovorans et Phanerochaete chrysosporium, confirmant leur potentiel technologique en condition aérobie. En condition anaérobie, une communauté microbienne présentant la capacité de dégrader les PCB a été développé. Une étude de la diversité du gène ADNr 16S au sein de cette communauté a permis d'identifier les espèces présentes dans cette communauté. Une analyse de cycle de vie évalue les performances environnementales de deux procédés de traitement de sols contaminés par des PCB, l'un thermique, l'autre biologique. Cette analyse permet de quantifier l’avantage environnemental du procédé biologique sur son concurrent thermique. / Soil is a complex interface between all compartments of the environment. Their pollution contributes to the spread of many pollutants. PCBs are persistent toxic compounds in the environment. Widely used especially in dielectric oils, they now contaminate many industrial floors. Heat treatment of these soils is very expensive and can cause the emission of dioxins. The objective of this work is to study a biological treatment process for the degradation of PCBs in soils. Biological degradation of PCBs has been observed in the presence of two cultured organisms, Burkholderia xenovorans and Phanerochaete chrysosporium, confirming their technological potential under aerobic conditions. Under anaerobic conditions, a microbial community with the ability to degrade PCBs was developed. A study of the diversity of 16S rDNA gene within this community has identified the species in this community. An analysis of life cycle assess the environmental performance of two methods for treating soils contaminated with PCBs, one thermal and one biological. This analysis quantifies the environmental benefit of the biological process compared with the heat treatment

To gain a better understanding of the mechanisms by which the white-rot decay fungus Phanerochaete chrysosporium regulates selective delignification versus simultaneous decay, differential gene expression of its two key enzymes were measured over the course of aspen and birch wood decay tests. The type of decay was determined by differential staining and scanning electron microscopy. Real-time qPCR assessed gene expression of four lignin peroxidase genes and two manganese peroxidase genes at each stage of decay. Differential expression was significant in the mnp2 gene between aspen and birch decay tests during incipient decay; abundant expression of mnp2 in aspen corresponded to early initiation of selective delignification, whereas birch underwent initial simultaneous decay in the absence of abundant mnp2 expression. The lipC gene was the most abundantly expressed lip gene at all time points in both wood species and likely plays an important role in regulating wood decay.

Dans les écosystèmes forestiers, la décomposition du bois est un processus majeur, notamment impliqué dans le cycle du carbone et des nutriments. Les champignons basidiomycètes saprotrophes, incluant les pourritures blanches, sont les principaux agents de cette décomposition dans les forêts tempérées. Bien que peu étudiées, des communautés bactériennes sont également présentes dans le bois en décomposition et cohabitent avec ces communautés fongiques. L'impact des interactions bactéries-champignons sur le fonctionnement d'une niche écologique a été décrit dans de nombreux environnements. Cependant, leur rôle dans le processus de décomposition du bois n'a été que très peu investigué. A partir d'expériences en microcosme et en utilisant une approche non cultivable, il a été démontré que la présence du champignon Phanerochaete chrysosporium influençait significativement la structure et la diversité des communautés bactériennes associées au processus de décomposition du hêtre (Fagus sylvatica). Par une approche cultivable, cet effet mycosphère a été confirmé, se traduisant par une augmentation de la densité des communautés bactériennes en présence du champignon ainsi que par une modification de la diversité fonctionnelle de ces communautés. Enfin, une approche polyphasique a été développée, combinant l'analyse des propriétés physico-chimiques du bois et des activités enzymatiques extracellulaires. Les résultats de cette expérience ont révélé que l'association de P. chrysosporium avec une communauté bactérienne issue de la mycosphère de ce dernier aboutissait à une dégradation plus importante du matériau bois par rapport à la dégradation par le champignon seul, démontrant pour la première fois des interactions bactéries-champignons synergiques dans le bois en décomposition / Wood decomposition is an important process in forest ecosystems in terms of their carbon and nutrient cycles. In temperate forests, saprotrophic basidiomycetes such as white-rot fungi are the main wood decomposers. While they have been less studied, bacterial communities also colonise decaying wood and coexist with these fungal communities. Although the impact of bacterial-fungal interactions on niche functioning has been highlighted in a wide range of environments, little is known about their role in wood decay. Based on microcosm experiments and using a culture-independent approach, we showed that the presence of the white-rot fungus Phanerochaete chrysosporium significantly modified the structure and diversity of the bacterial communities associated with the degradation of beech wood (Fagus sylvatica). Using a culture-dependent approach, it was confirmed that in the presence of the fungus the mycosphere effect resulted in increased bacterial abundance and modified the functional diversity of the fungal-associated bacterial communities. Lastly, a polyphasic approach simultaneously analysing wood physicochemical properties and extracellular enzyme activities was developed. This approach revealed that P. chrysosporium associated with a bacterial community isolated from its mycosphere was more efficient in degrading wood compared to the fungus on its own, highlighting for the first time synergistic bacterial-fungal interactions in decaying wood

Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus Phanerochaete carnosa has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by P. carnosa, its genome was sequenced and transcriptomes were evaluated after growth on wood compared to liquid medium. Results indicate that P. carnosa differs from P. chrysosporium in the number and expression levels of genes that encode lignin peroxidase (LiP) and manganese peroxidase (MnP), two enzymes that modify lignin present in wood. P. carnosa has more genes for MnP with higher expression levels than LiP, while the reverse has been observed for P. chrysosporium. The abundances of transcripts predicted to encode lignocellulose-modifying enzymes were then measured over the course of P. carnosa cultivation on four wood species. Profiles were consistent with decay of lignin before carbohydrates. Transcripts encoding MnP were highly abundant, and those encoding MnP and LiP featured significant substrate-dependent response. Since differences in modes of lignin degradation catalyzed by MnP and LiP could affect the ability of each to degrade lignin from different types of wood, their activity on various hardwoods and softwoods were tested. Results suggest that MnP degrades softwood lignin more effectively than hardwood lignin, consistent with high levels of this enzyme in P. carnosa.

Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus Phanerochaete carnosa has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by P. carnosa, its genome was sequenced and transcriptomes were evaluated after growth on wood compared to liquid medium. Results indicate that P. carnosa differs from P. chrysosporium in the number and expression levels of genes that encode lignin peroxidase (LiP) and manganese peroxidase (MnP), two enzymes that modify lignin present in wood. P. carnosa has more genes for MnP with higher expression levels than LiP, while the reverse has been observed for P. chrysosporium. The abundances of transcripts predicted to encode lignocellulose-modifying enzymes were then measured over the course of P. carnosa cultivation on four wood species. Profiles were consistent with decay of lignin before carbohydrates. Transcripts encoding MnP were highly abundant, and those encoding MnP and LiP featured significant substrate-dependent response. Since differences in modes of lignin degradation catalyzed by MnP and LiP could affect the ability of each to degrade lignin from different types of wood, their activity on various hardwoods and softwoods were tested. Results suggest that MnP degrades softwood lignin more effectively than hardwood lignin, consistent with high levels of this enzyme in P. carnosa.

In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs). The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work. The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins. Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.

This diploma thesis is focused on study of potential production of extracellular hydrolytic enzymes. The theoretical part deals with characterization of selected hydrolytic enzymes, their catalytic properties, the possibility of extracellular hydrolase production by fungi and their applications. In experimental part production strains Aureobasidium pullulans, Fusarium solani and Phanerochaete chrysosporium were used. Productions of cellulase, amylase, xylanase, lipase, protease and lignin-degraded enzymes (laccase, manganese- dependent peroxidase, lignin peroxidase) were observed. Cultivations were carried out in submersed mode in mineral medium supplemented by waste co-substrates such as wheat bran, corn bran, rice bran and oat bran, sawdust, rice, apple fiber, egg pasta and egg-free pasta. Production of enzymes depended on the substrate type and time of cultivation. The highest cellulase, xylanase and amylase activities were measured in the first period of cultivation (3 to 7 day). Lignin-degraded enzymes and proteases were produced at the end of cultivation (7 to 10 days). Lipolytic activity was detected only in A. pullulans, where the activity increased with time of cultivation. The highest value was determined during cultivation on wheat bran (3.6 nmol/ml.min). The highest xylanase and celulase activity (170.3 nmol/ml.min, 248.0 nmol/ml.min) were determined during cultivation of F. solani on corn bran. The highest amylase activity (111.8 nmol/ml.min) was reported in P. chrysosporium during the cultivation on rice. The highest protease activity (68.0 nmol/ml.min) was determined in F. solani grown on wheat bran. The best producer of laccase was A. pullulans, the highest production was recorded for egg-free pasta (27.0 nmol/ml.min). The maximum lignin peroxidase activity (12.5 nmol/ml.min) was measured during the cultivation of F. solani on egg pasta, while the highest yield of Mn-dependent peroxidase (7.7 nmol/ml.min) was achieved during the cultivation of A. pullulans on wheat bran. Lignin-degraded enzymes behaved as inductive, while the other enzymes were produced in mineral medium too. Activity of cellulase in the mineral medium was in A. pullulans strain higher than in media with waste substrates. Enzymes produced into A. pullulans medium were purified by ultrafiltration, ion exchange chromatography and gel filtration.