Supercharging of a family-5 glycoside hydrolase Cel5A from Thermobifida fusca to improve cellulose hydrolysis
Creators: name: De Chellis, Antonio
Publisher: name: No Publisher Supplied
Year: 2022

description: The feasibility of producing useful fuels and chemicals from lignocellulosic biomass hinges on the efficiency of biomass deconstruction catalyzed by different enzymes such as cellulases and hemicellulases. Enzymatic hydrolysis is hindered by biomass recalcitrance caused by the tight crystalline cellulose structure as well as the structural organic biopolymer lignin. Lignin forms a protective covering over core cellulose and hemicellulose restricting access to enzymes while also being a major source of irreversible non-productive binding. Thermochemical pretreatment is often used to disrupt the lignin coating in order to increase exposure of cellulose and hemicellulose, but delocalized lignin remains present and still available for irreversible binding of cellulases/hemicelluases. As such, current industrial processes require high enzyme loadings to supplement low activity on crystalline cellulose, as well as enzyme lost to non-productive binding. Engineering strategies, such as enzyme supercharging which refers to the process of introducing positive or negatively charged amino acids in solvent exposed areas to create highly negative/positive net charge, have been useful in the past to combat biomass recalcitrance. Past work has focused on improving thermal stability of cellulases, as well as altering surface interactions in order to reduce binding to lignin but has not exhibited higher activity on highly crystalline cellulose. This thesis focuses on a supercharged library consisting of a family 5 glycoside hydrolase (GH5) Cel5A and its native family 2a carbohydrate binding module CBM2a from the thermophilic microbe Thermobifida fusca. The library consists of 33 enzymes in total, including the wild-type (WT), with mutations being made on both the CBM2a binding module and Cel5A catalytic domain (CD) spanning a net charge range of -51.3 to 36.9. To gather preliminary activity data on the entire library, each design was expressed in E. coli as small-scale cultures, and the cell lysate was screened using numerous assays. Activity data was collected using several substrates including soluble para-nitrophenol cellobioside (pNP-C), pretreated ammonia fiber expansion (AFEX) corn stover biomass, and crystalline cellulose. Lysate screening enabled the reduction of the library size from 33 mutants in total to 16 that showed activity either similar or greater compared to the wild type. From this reduced library, four constructs with CBM and two constructs with CD mutations were expressed and purified on a large scale. The purified mutants were used to validate the cell lysate screening and characterized further. Three notable mutants, one negatively supercharged, and two positively supercharged stood out compared to the wild-type enzyme. All three constructs have exhibited up to a 2-fold increase in activity on crystalline cellulose-I, and both positively supercharged enzymes have shown a five degree increase in their optimal hydrolysis temperature. Overall, this work provides insights on how supercharging can be used to tune surface interactions to decrease non-productive binding, increase activity on crystalline cellulose, as well as show how supercharging can impact thermal resilience of cellulase enzymes.