Characterization of in vitro stability for two processive endoglucanases as exogenous fibre biocatalysts in pig nutrition

Development of highly efficacious exogenous fibre degradation enzymes can enhance efficiency of dietary fibre utilization and sustainability of global pork production. The objectives of this study were to investigate in vitro stability for two processive endoglucanases, referred to as GH5-tCel5A1 and GH5-p4818Cel5_2A that were overexpressed in CLEARCOLIBL21(DE3). Three-dimensional models predicted presence of Cys residues on the catalytic site surfaces of GH5-tCel5A1 and GH5-p4818Cel5_2A; and time course experimental results shown that both cellulases were susceptible to auto-oxidation by airborne O(2) and were unstable. Furthermore, we examined these endoglucanases’ stability under the mimicked in vitro porcine gastric and the small intestinal pH and proteases’ conditions. Eadie-Hofstee inhibition kinetic analyses showed that GH5-tCel5A1 and GH5-p4818Cel5_2A respectively lost 18 and 68% of their initial activities after 2-h incubations under the gastric conditions and then lost more than 90% of their initial activities after 2–3 h of incubations under the small intestinal conditions. Therefore, further enzyme protein engineering to improve resistance and alternatively post-fermentation enzyme processing such as coating to bypass the gastric-small intestinal environment will be required to enable these two processive endoglucanases as efficacious exogenous fibre enzymes in pig nutrition application.