Role of cysteine residues in thermal inactivation of fungal Cel6A cellobiohydrolases

Numerous protein engineering studies have focused on increasing the thermostability of fungal cellulases to improve production of fuels and chemicals from lignocellulosic feedstocks. However, the engineered enzymes still undergo thermal inactivation at temperatures well below the inactivation temperatures of hyperthermophilic cellulases. In this report, we investigated the role of free cysteines in the thermal inactivation of wild-type and engineered fungal family 6 cellobiohydrolases (Cel6A). The mechanism of thermal inactivation of Cel6A is consistent with disulfide bond degradation and thiol-disulfide exchange. Circular dichroism spectroscopy revealed that a thermostable variant lacking free cysteines refolds to a native-like structure and retains activity after heat treatment over the pH range 5-9. Whereas conserved disulfide bonds are essential for retaining activity after heat treatment, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A as well as Cel6A from Hypocrea jecorina and Humicola insolens.