|dc.description.abstract||Microbes are fast becoming the solution to the energy crisis. Alternative fuels that can be produced by microbes include bioethanol, biodiesel, methane and biohydrogen. This study aimed to identify and characterize a thermostable endoglucanase, a carbohydrate active enzyme which would be part of a cocktail of enzymes used to breakdown cellulose to liberate glucose. The glucose in turn can be fermented to produce bioethanol.
Twenty seven fungal isolates were screened for endoglucanase activity on agar plates supplemented with 1% carboxymethyl cellulose. Zones of hydrolysis were visualized by Congo red staining. Ten isolates were selected based on the diameters of hydrolytic zones (presenting high endoglucanase activity) for time course analysis for optimal production of endoglucanase. Isolate D9 (Aspergillus fumigatus) was found to be the highest producer and time course analysis revealed that the highest production is achieved on Day 9 after inoculation. The crude enzyme extract was produced in bulk and precipitated in 40 - 60% ammonium sulphate in order to remove unwanted proteins and to concentrate the endoglucanases. However, following dialysis, more than 80% proteins and endoglucanase activity was lost. Other avenues such as IEF fractionation and freeze drying were explored to achieve partial purification and concentration, respectively, but these proved fruitless.
Thus the focus of the study shifted to cloning, heterologously expressing and characterizing an endoglucanase from Phialophora alba, a fungus known to produce lignocellulosic enzymes. Whole genome sequencing was performed and open reading frames (ORFs) were identified using the CLC Main Workbench. These ORFs were analyzed using the CAZymes Analysis Toolkit to identify carbohydrate active enzyme genes. A single endoglucanase gene (GH5) was identified and selected for the duration of the study. Primer sets GH5_ORF_F; GH5_ORF_R and Exp_GH5_ORF_F; Exp_GH5_ORF_R, were specifically designed for cloning into plasmids pTZ57R/T and pPIC9, respectively. The recombinant plasmid pPIC9_13_Eg_GH5 was successfully integrated into Pichia pastoris GS115 genome. The heterologous enzyme was produced; however, no endoglucanase activity was detected. The gene was expressed in Escherichia coli BL21 (DE3), however, no endoglucanase activity was detected in this instance as well. Bioinformatic analysis of the CAZome of P. alba shows a preference for xylan and chitin over cellulose. P. alba appears to be a poor cellulose degrader possessing only a single endoglucanase gene, however, it does possess auxillary activity enzymes enzymes that may also be involved in the breakdown on cellulose. This therefore provides insight into why this enzyme could be inactive as well as sheds light on the plant polysaccharide degradative abilities of this fungus.||en_US