PROTEIN ENGINEERING STUDIES ON BACILLUS THERMOCATENULATUS LIPASE
Biological Sciences and Bioengineering, PhD Thesis, 2012
Assoc. Prof. Ugur Sezerman (Thesis Supervisor), Prof. Dr. Serdar Kuyucak, Prof. Dr. Selim Çetiner, Asst. Prof. Alpay Taralp, Assoc. Prof. Levent Öztürk
Date & Time: October 15th, 2012 - 10:00
Place: FENS L056
Keywords: Protein engineering, Lipase, Selectivity, Stability
Bacillus thermocatenulatus lipase (BTL2) is a thermostable enzyme with distinct tributyrin selectivity and currently two different resolved structures are publicly available representing the; a) active-monomer form (PDB ID: 2w22) and b) inactive-dimer form (PDB ID: 1KU0). Using the information gained from these two structures, here we report the results of two case studies related to the protein engineering of BTL2 aiming to improve its chain-length selectivity and to assess the impact of the lid tryptophanes. For both of these studies, the experimental approach was to generate specific BTL2 mutants, which we have implemented using site-directed mutagenesis, heterologous protein expression and purification methodologies.
In the first study, a rational design of BTL2 is carried out to lower the activation barrier of hydrolysis of short chain substrates. The results indicate that the mutant L360F optimizes the physical and chemical interactions in BTL2 to lower the activation barrier for C4 and elevate it for C8 hydrolysis. For the second case, a semi-rational design is implemented to investigate the impacts of the two tryptophanes (W212, W235) in the lid domain of BTL2. Particularly, W212A is resulted in the severe loss of the thermostability and aggregation but also in the improvement of zinc tolerance. In this sense, we envision that the aggregation of BTL2 is mediated through a zinc domain and W212 is a secondary but vital residue for this action.