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dc.contributor.authorAuta, Richard
dc.date.accessioned2016-04-25T09:20:34Zen
dc.date.available2016-04-25T09:20:34Zen
dc.date.issued2015-11
dc.identifier.urihttp://hdl.handle.net/2436/606935
dc.descriptionA thesis submitted.....
dc.description.abstractβ-glucosidases are important components of the cellulase enzyme system in which they not only hydrolyse cellobiose to glucose, but also remove the feedback inhibition effects of cellobiose on exoglucanase and endoglucanase thereby increasing the rate of cellulose degradation to fermentable sugars. A total of 166 proteins were identified as β-glucosidases after manual BLASTp search on the Aspergillus comparative database from eight species. Evidence for Horizontal Gene Transfer (HGT) of bacterial origin of some β-glucosidase genes was provided by their lack of introns, absence of some fungal specific amino acid insertions in their sequences and unusual positions in phylogenetic trees showing similarities to bacterial proteins. A rapid plate assay based on Congo red methods was developed to study the optimum parameters such as pH and temperature for growth of strains and activities of the enzymes produced. Bacterial cellulose (BC) was produced by Gluconacetobacter xylinus. For the first time a fully detailed characterization by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), Differential scanning calorimeter (DSC), Thermogravimetric analysis (TGA) and 13Carbon Solid State Nuclear Magnetic Resonance (SSNMR) of pure BC before and after treatment with a commercially available Aspergillus cellulase enzyme was demonstrated. Two encoding sequences for novel Aspergillus nidulans hydrophobin genes ANID_05290.1 and ANID_07327 that do not fall into either the class I or class II category of hydrophobins were successfully cloned. Two encoding sequences for a novel β-glucosidase gene from an Aspergillus niger strain from Nigeria were amplified and cloned from genomic DNA using PCR. Aspergillus nidulans β-glucosidases (AN2227 and AN1804) expressed in Pichia were purified to homogeneity by using ammonium sulphate precipitation and DEAE-Sephadex A-50 chromatography. Both enzymes had a remarkably broad pH and temperature profile. Further experiments on the development of a technology for lignocellulose degradation based on co-production of β-glucosidase with hydrophobin for biofuel production are suggested.
dc.language.isoen
dc.subjectβ-glucosidase
dc.subjectBioinformatics
dc.subjectAspergillus
dc.subjectHorizontal Gene Transfer
dc.subjectpara-Nitrophenyl β-D-glucopyranoside
dc.subjectBacterial cellulose
dc.subjectscanning electron microscopy
dc.subjectX-Ray Diffraction
dc.subjectGluconacetobacter xylinus
dc.subjectFourier Transform Infrared Spectroscopy
dc.titlePURIFICATION AND CHARACTERISATION OF NOVEL RECOMBINANT β-GLUCOSIDASES FROM ASPERGILLUS WITH APPLICATION IN BIOFUEL PRODUCTION
dc.typeThesis or dissertation
refterms.dateFOA2018-08-20T16:22:43Z
html.description.abstractβ-glucosidases are important components of the cellulase enzyme system in which they not only hydrolyse cellobiose to glucose, but also remove the feedback inhibition effects of cellobiose on exoglucanase and endoglucanase thereby increasing the rate of cellulose degradation to fermentable sugars. A total of 166 proteins were identified as β-glucosidases after manual BLASTp search on the Aspergillus comparative database from eight species. Evidence for Horizontal Gene Transfer (HGT) of bacterial origin of some β-glucosidase genes was provided by their lack of introns, absence of some fungal specific amino acid insertions in their sequences and unusual positions in phylogenetic trees showing similarities to bacterial proteins. A rapid plate assay based on Congo red methods was developed to study the optimum parameters such as pH and temperature for growth of strains and activities of the enzymes produced. Bacterial cellulose (BC) was produced by Gluconacetobacter xylinus. For the first time a fully detailed characterization by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), Differential scanning calorimeter (DSC), Thermogravimetric analysis (TGA) and 13Carbon Solid State Nuclear Magnetic Resonance (SSNMR) of pure BC before and after treatment with a commercially available Aspergillus cellulase enzyme was demonstrated. Two encoding sequences for novel Aspergillus nidulans hydrophobin genes ANID_05290.1 and ANID_07327 that do not fall into either the class I or class II category of hydrophobins were successfully cloned. Two encoding sequences for a novel β-glucosidase gene from an Aspergillus niger strain from Nigeria were amplified and cloned from genomic DNA using PCR. Aspergillus nidulans β-glucosidases (AN2227 and AN1804) expressed in Pichia were purified to homogeneity by using ammonium sulphate precipitation and DEAE-Sephadex A-50 chromatography. Both enzymes had a remarkably broad pH and temperature profile. Further experiments on the development of a technology for lignocellulose degradation based on co-production of β-glucosidase with hydrophobin for biofuel production are suggested.


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