PURIFICATION AND CHARACTERISATION OF NOVEL RECOMBINANT β-GLUCOSIDASES FROM ASPERGILLUS WITH APPLICATION IN BIOFUEL PRODUCTION

2.50
Hdl Handle:
http://hdl.handle.net/2436/606935
Title:
PURIFICATION AND CHARACTERISATION OF NOVEL RECOMBINANT β-GLUCOSIDASES FROM ASPERGILLUS WITH APPLICATION IN BIOFUEL PRODUCTION
Authors:
Auta, Richard
Abstract:
β-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.
Issue Date:
Nov-2015
URI:
http://hdl.handle.net/2436/606935
Type:
Thesis
Language:
en
Description:
A thesis submitted.....
Appears in Collections:
E-Theses

Full metadata record

DC FieldValue Language
dc.contributor.authorAuta, Richarden
dc.date.accessioned2016-04-25T09:20:34Zen
dc.date.available2016-04-25T09:20:34Zen
dc.date.issued2015-11en
dc.identifier.urihttp://hdl.handle.net/2436/606935en
dc.descriptionA thesis submitted.....en
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.en
dc.language.isoenen
dc.subjectβ-glucosidaseen
dc.subjectBioinformaticsen
dc.subjectAspergillusen
dc.subjectHorizontal Gene Transferen
dc.subjectpara-Nitrophenyl β-D-glucopyranosideen
dc.subjectBacterial celluloseen
dc.subjectscanning electron microscopyen
dc.subjectX-Ray Diffractionen
dc.subjectGluconacetobacter xylinusen
dc.subjectFourier Transform Infrared Spectroscopyen
dc.titlePURIFICATION AND CHARACTERISATION OF NOVEL RECOMBINANT β-GLUCOSIDASES FROM ASPERGILLUS WITH APPLICATION IN BIOFUEL PRODUCTIONen
dc.typeThesisen
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