Laurence Cheng's MSc Defence

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Poor efficiency of dietary fibre utilization limits pork production profit margins and environmental sus-tainability. This thesis research was to investigate stability of two processive endoglucanases, referred to as GH5-tCel5A1 and GH5-p4818Cel5_2A and overexpressed in the ClearColi® BL21 (DE3) cell. Three-dimensional models predicted presence of cysteine residues on the cellulases’ catalytic sites; and results of in vitro time course experiments further shown these cellulases were susceptible to auto-oxidation by air-borne O2 and were unstable. In vitro cellulases’ stability was also examined under the mimicked porcine gastric and the small intestinal conditions. Eadie-Hofstee inhibition kinetic analyses shown that both cellulases lost significant levels of their initial enzyme activities within 2 – 3 h of incu-bations and were thus not stable under the porcine gastrointestinal environmental conditions. It is con-cluded that enzyme protein engineering and alternatively post-fermentation enzyme processing need to be further pursued to enable both cellulases as potentially efficacious exogenous fibre enzymes.

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