Pillai, Santhosh Kumar KuttanAmobonye, AyodejiMagwaza, Buka Welcome2025-07-212025-07-212025-05https://hdl.handle.net/10321/6101Submitted in fulfilment for the Degree of Master of Applied Science in Biotechnology, Durban University of Technology, Durban, South Africa, 2025.Beauveria bassiana is a popular entomopathogenic fungal endophyte that is used industrially as a biocontrol agent. It has been noted to be non-pathogenic to humans, animals and plants and their ability to utilise various agro-residues for its metabolism has been exploited for the production of some lignocellulosic enzymes. Previous studies have mostly focused on the production of key lignocellullose hydrolysing enzymes, however, little is known about the ability of B. bassiana to produce accessory enzymes such as β-glucosidase which also aid in lignocellullose breakdown. Hence, this study was aimed at investigating the production, the biochemical characteristics, and the potential application of a β-glucosidase from the strain designated as Beauveria bassiana SAN01. For these aims to be achieved, the production parameters of β-glucosidase were optimised using a statistical approach. Furthermore, to enhance the evaluation of the biochemical properties and potential industrial application of the enzyme, it was purified to homogeneity using salt precipitation, and chromatography. The preliminary screening of seven agricultural residues showed that the haulm of Bambara an underutilised African legume- supported the highest β-glucosidase production, hence, statistical optimisation of enzyme production was performed using this biomass as the sole carbon source. The three-level statistical optimisation experiments resulted in a ~5.36-fold increase in β-glucosidase production from the unoptimised level of 132.71 U/mL to 711 U/mL, under optimal conditions (Bambara - 57 g/L, KCl - 302 mg/L, NaCl -154 mg/L, agitation -150 rpm, and incubation time - 223 h). Ammonium sulphate precipitation followed by dialysis and gel filtration chromatography were used to purify β-glucosidase produced by B. bassiana SAN01 to homogeneity. The purified β-glucosidase was demonstrated to have a specific activity of 496 U/mg and a molecular mass of ~116 kDa by SDS-PAGE; its activity pattern was also confirmed via in-gel zymography using 4-methylumbelliferyl-β-D-glucopyranoside as the substrate. The enzyme activity was recorded to be optimal at pH 5.0 and 60°C and the enzyme also displayed significant thermal stability from temperatures 30-50°C, retaining almost 60% of its activity at 50°C after 4 h of incubation. Subsequently, the potential of B. bassiana SAN01 β-glucosidase as an accessory enzyme in lignocellulose saccharification was demonstrated by its effectiveness in the hydrolysis of cellobiose converting more than 90% of the substrate to glucose. Finally, some structural insights were gained into the enzyme using a computational approach. The in silico prediction of the enzyme revealed that it has an isoelectric point of 5.59, that it was hydrophilic and thermostable. The modelled 3D structure of B. bassiana β-glucosidase confirmed that it belongs to the GH 1 family and the model was validated by the presence of ~ 96% of its amino acid residues in the favoured region of the Ramachandran plot. The docking of the enzyme with cellobiose and 4-nitrophenol β-D glucopyranoside demonstrated the significant affinity of both substrates to the enzyme while revealing its most probable active site. Results from this study demonstrate B. bassiana as a hyper-producer of β-glucosidase as the production level in this study is one of the highest ever recorded for an entomopathogenic fungi; thus the study highlights the immense potential of B. bassiana in the processing of lignocellulosic biomass to biofuels.147 penBeauveria bassiana SAN01β-glucosidaseLignocellulosic enzymesBiomassEndophytic fungiEntomopathogenic fungiBiological pest control agentsGlucosidasesThesishttps://doi.org/10.51415/10321/6101