PhD Dissertation: Aslı Yenenler
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PROTEIN ENGINEERING APPLICATIONS FOR FUNCTIONAL ENHANCEMENT OF CELLULASE AND LIPASE ENZYMES

 

ASLI YENENLER

BIO, PhD Dissertation, 2017

 

Thesis Jury

Prof. Dr. İsmail Çakmak (Thesis Advisor), Prof. Dr. Osman Uğur Sezerman

, Assoc. Prof. Devrim Gözüaçık, Assoc. Prof. Levent Öztürk,

Prof. Dr. Dilek Kazan

 

Date & Time: January 06th, 2017 –  9.40 AM

Place: FENS L065

Keywords : Protein engineering, Lipase, Cellulase, DNA shuffling, Domain swapping, MD simulation

 

Abstract

 

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 limited enzymatic activity of lipase in organic solvents. In 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. DNA shuffling method enables us to track the molecular evolution of cellulase genes in laboratory environment. In second part, we engineered EGI endoglucanase enzyme via domain swapping of Co2+ coordination 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. Our results suggested that the structural characteristics of CBHI were transferred to EGI enzyme that would increase its feasibility in harsh conditions of biotechnological applications. 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. In general, the structural restrictions and conformational stability problems of BTL2 in toluene were reported, and we described how these problems limit the usage of BTL2 in organic solvent. Among many regions, Val198-Pro218 was reported as the most suffered region and several mutations were suggested to solve the problems about conformational rigidity of BTL2 in toluene. Indeed, a theoretical basis about the behavior of BTL2 in toluene was constituted for further studies, aiming to provide the improvements of BTL2’s usage in hydrophobic media.