Protein engineering applications for functional enhancement of cellulase and lipase enzymes
Yenenler, Aslı (2017) Protein engineering applications for functional enhancement of cellulase and lipase enzymes. [Thesis]
Cellulases and lipases are considered as metabolic enzymes and widely used in industrial applications. Due to the growing demand of their usage in harsh conditions of industry, there exists a need for improvements in their catalytic properties with protein engineering methods. In this thesis, we applied several protein-engineering methods to generate novel cellulase enzymes with altered enzyme kinetics and to understand the main reasons behind the limited enzymatic activity of lipase in organic solvents. In the first part, we generated two novel cellulase enzymes (EG3_S1&EG3_S2) through DNA shuffling and reported 15.6-fold improvements in enzymatic activity of EG3_S2 (at 45oC) with respect to native. Also, EG3_S2 displayed the greater thermal stability and it is considered as a better biocatalyst than native with ~12.8-fold and ~6.5-fold improvements in Vmax and kcat/km, respectively. In the second part, we engineered EGI endoglucanase enzyme via domain swapping of Co2+ region from CBHI. Here, a new endoglucanase, named as EGI_swapped was reported with modified structural and enzymatic properties. It displays ~1.7-fold better thermal stability than native without compromising the catalytic efficiency. Lastly, we performed molecular dynamic simulations with BTL2 lipase in toluene environment to provide an understanding how toluene molecules impose the structural restriction on BTL2. Val198-Pro218 was reported as the most suffered region and several mutations were suggested to solve the problems about the conformational stability of BTL2 in toluene. Indeed, a theoretical basis for the behavior of BTL2 in toluene was constituted for further studies, aiming to provide the improvements of BTL2's usage in hydrophobic media.
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