No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Thesis (PhD. (Microbiology)) --University of Limpopo, 2013 / The current study investigated four fungal species namely Aspergillus niger FGSC A733, Aspergillus versicolor EF23, Penicillium citrinum AZ01 and Trichoderma harzianum NCGR 0509 for their abilities to produce cellulases and xylanases in submerged and solid state fermentations. Five different substrates (carboxymethyl cellulose, xylan, common thatch grass, wheat bran and Jatropha curcas seed cake) were examined for their potential use as low cost feedstock for fermentation by the fungal species. Aspergillus niger FGSC A733 produced the highest titres of cellulase and xylanase in solid state fermentations using wheat bran as a substrate. However, because of the need to lower the cost of enzyme production, Jatropha seed cake a relatively underutilised oilseed cake was used. Supplementation of the Jatropha seedcake with 10% common thatch grass (Hyperrhenia sp) resulted in a fivefold increase in the levels of xylanase produced. Cellulase production was not affected by this supplementation. Addition of ammonium chloride increased production of xylanase while cellulase production was not affected nitrogen supplementation. Maximum xylanase was produced on Jatropha seed cake at 25 °C after 96 hours while cellulase was maximally produced at 40 °C after 96 hours of solid state fermentations. Peak production of xylanase was obtained at an initial pH of 3 whilst cellulase was maximally produced at an initial pH of 5. The crude xylanase was most active at pH 5 and cellulase at pH 4. The optimum temperature for cellulase activity was 65 °C and that of xylanase was 50 °C. Under optimized conditions, 6087 U/g and 3974 U/g of xylanase and cellulase per gram of substrate used were obtained respectively. The diversity of cellulases was investigated so as to determine the most appropriate enzyme mixture for saccharification of the common thatch grass. Proteins from the four species under investigation were partially purified by affinity chromatography on swollen Avicel. The proteins were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDS-PAGE and zymography. Potential cellulase bands from SDS-PAGE were sequenced by mass spectrometry. The basic logical alignment tool (BLAST) and Clustal W were used for matching and identifying the sequences with closely related ones in the databases. The identified proteins from Penicillium citrinum AZ01 and Aspergillus versicolor EF23 were found to closely resemble a catalytic domain of cellobiohydrolase from Trichoderma sp. The three proteins obtained from Aspergillus niger showed resemblance to 1,4-beta glucan cellobiohydrolase A precursor from Aspergillus niger FGSC A733 was also found to have cellobiase and endoglucanase activity was determined using cellobiase and carboxymethyl cellulose as substrates. Cellulase and xylanase zymograms of proteins from A. niger FGSC A733 demonstrated six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase. The cellulase produced by Aspergillus niger FGSC A733 on Jatropha seed cake under optimised conditions was used for saccharification of 2% (w/v) common thatch grass (CTG) in combination with Celluclast™. Celluclast™ and Aspergillus niger cellulase were mixed at different ratios and the amount of glucose produced over time was monitored using high performance liquid chromatography (HPLC). A ratio of 2 volumes Celluclast™ to one volume Aspergillus niger cellulase was chosen for the saccharification process. The main enzymes in the mixture were identified using peptide mass fingerprinting as endoglucanases from the Celluclast™ and cellobiase from the Aspergillus niger cellulase. Concentration of the Celluclast™ tenfold times (164 FPU) improved the yield of glucose by 42.8 and 37.8% in acid and alkali pre-treated CTG, respectively. Concentrating Aspergillus niger cellulase (13.2 FPU) decreased the production of glucose by 4.8% in acid pre-treated CTG while in alkali pre-treated CTG, a 5% increase in glucose production was observed. Increasing the substrate loading of acid pre-treated CTG from 2% to 10% (w/v) resulted in a two and a half times increase in glucose production while an increase of 1.5 g/l glucose was obtained from 7% (w/v) alkali pre-treated CTG. Addition of xylanases from Aspergillus niger to the Celluclast™-Aspergillus niger cellulase mixture decreased glucose production by 16.3% on acid pre-treated CTG while there was an increase of 18.3% glucose in alkali pre-treated CTG. Addition of enzyme preparations from Aspergillus versicolor EF23, Penicillium citrium AZ01 and Trichoderma harzianum NCGR 0509 to the Celluclast™-Aspergillus niger cellulase mixture resulted in lower glucose production both in acid and alkali pre-treated CTG. Addition of Pentopan™ improved glucose production by 8 and 25% on 10% acid and 7.5% alkali loading of pre-treated CTG respectively. The optimal conditions for the production of the glucose rich hydrolysate in 10% (w/v) acid and 7% (w/v) alkali pre-treated CTG was found to be the use of Celluclast™-Aspergillus niger cellulase-Pentopan™ mixture (164 FPU Celluclast™ and 13 FPU Aspergillus niger cellulase, 7178 IU) Pentopan™ at 50 °C for 32 hours. The fermentability of the glucose in glucose-rich CTG hydrolysates to ethanol using Saccharomyces cerevisae WBSA 1386 and Candida shehatae CSIR Y-0492 was investigated. The highest yield of ethanol produced by S. cerevisae WBSA 1386 was 9.8 g/l in the alkali pre-treated CTG hydrolysate and 8.7 g/l in acid pre-treated CTG. C. shehatae CSIR Y-0492 produced 9 g/l of ethanol in alkali pre-treated CTG within 48 hours while acid pre-treated CTG hydrolysate produced 8.8 g/l of ethanol within 24 hours of the fermentation process. Addition of the nutrient supplement boosted the ethanol yield in the acid pre-treated hydrolysates. The consumption of glucose during fermentation by S. cerevisae WBSA 1386 and C. shehatae CSIR Y-0492 on average was 97%. The C. shehatae CSIR Y-0492 was expected to produce much higher ethanol yield than the Saccharomyces because of its ability to utilize xylose for ethanol production. This however was not observed in this investigation. The conclusion of this study is that it is possible to produce bioethanol from Hyperrhenia spp. (CTG) using a combination of fungal enzymes for the production of fermentable sugars.

Les hélicases à ARN de la famille DEAH/RHA sont impliquées dans la plupart des processus essentiels à la vie tels que l'épissage, la biogenèse des ribosomes, la réplication, la transcription ou encore la détection d’ARN viraux. Ces enzymes sont capables de catalyser la dissociation de duplexes d'ARN, la réorganisation de structures secondaires ou de remodeler des complexes ARN-protéines. L'hélicase DEAH/RHA Prp43 présente la particularité d'être bifonctionnelle. Prp43 est impliquée dans l'épissage des Pré-ARNm, où elle assure le recyclage du spliceosome et du lasso, mais aussi dans la biogenèse des ribosomes où elle est impliquée dans la maturation des deux sous-unités. Prp43 est activée et régulée par cinq partenaires protéiques : Ntr1, Gno1, Pfa1, RBM5 et GPATCH2. Ces partenaires protéiques présentent tous un domaine G-patch et sont capables de stimuler les activités hélicase et ATPase de Prp43. La structure cristallographique de Prp43 en complexe avec l'ADP a été résolue au laboratoire. Cette structure a mis en évidence un mode de fixation du nucléotide inédit chez les autres hélicases, notamment au niveau de la base qui s'empile entre la phénylalanine 357 (F357) du domaine RecA2 et l'arginine 159 (R159) du domaine RecA1. Les déterminants de l'activation de Prp43 par les protéines à domaine G-patch demeurent méconnus. Dans ce travail, nous avons cherché à déterminer quel était le rôle de l’empilement de la base dans l’activation de Prp43. Nous présentons ici plusieurs structures cristallographiques de Prp43 en complexe avec tous les nucléotides diphosphates(NDP) et les désoxynucléotides triphosphates (dNDP). Ces structures ont permis de conclure qu'il y avait des différences dans l’empilement de la base selon le (d)NDP considéré. Des dosages d'activité NTPase de Prp43 avec et sans son partenaire protéique Pfa1 montrent que lorsque la base ne s'empile pas avec la F357 et la R159, l'activité de l'enzyme n'est pas correctement régulée par son partenaire protéique. Les dosages d’activité enzymatique sur les mutants ponctuels F357A et R159A révèlent que le résidu F357 permet de moduler l’activité de Prp43. Tous ces résultats nous ont permis de mettre en évidence un modèle de la régulation de Prp43 par les protéines à domaines G-patch et d'expliquer l'importance du mode de fixation de la base à l'enzyme dans cette régulation. / RNA helicases from the DEAH/RHA family are involved in most of essential processes of life such as pre-mRNA splicing, ribosome biogenesis, replication, transcription or viral RNA sensing. These enzymes are able to catalyze RNA unwinding, secondary structures reorganization or RNA-protein complexes remodeling. The DEAH/RHA helicase Prp43 is remarkable because it is bifunctional, as it is involved both in pre-mRNA splicing, where it is responsible of spliceosome and lariat recycling and in the biogenesis of the two ribosomal subunits. Prp43 is activated by five protein partners: Ntr1, Gno1, Pfa1, RBM5 and GPATCH2. These protein partners all possess a G-patch domain and are able to stimulate helicase and ATPase activity of Prp43. The structure of Prp43 in complex with ADP has been solved by X-ray crystallography. The structure reveals that the nucleotide is bound to the enzyme in a novel mode that has never been observed in other known helicase structures. The specific feature of this binding mode is the base, stacked between phenylalanine (F357) from RecA2 domain and an arginine (R159) from RecA1 domain. Features of the activation of Prp43 by G-patch proteins are unclear. In this work, we investigated the role of base stacking in the activation of Prp43. We present several structures of Prp43 bound to all the nucleotide diphosphates (NDP) and deoxynucleotide diphosphates (dNTP). These results indicate that there are differences in stacking according to the (d)NDP bound to the enzyme. NTPase activity assays revealed that when stacking is weakened, Prp43 activity cannot be properly regulated by its protein partner Pfa1. Moreover, point mutations F357A and R159A show that stacking of F357 permits to modulate Prp43 activity. All these results allow us to propose a model of NTPase activity activation of Prp43 by G-patch proteins and to highlight the importance of base stacking in this regulation.