Characterization of Processive Endoglucanase Stability in Vitro as an Exogenous Fibre Biocatalyst in Pig Nutrition
Poor efficiency of dietary fibre utilization limits pork production profit margins and environmental sustainability. 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 incubations and were thus not stable under the porcine gastrointestinal environmental conditions. It is concluded 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.