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Protein engineering studies on bacillus thermocatenulatus lipase

Durmaz, Emel (2012) Protein engineering studies on bacillus thermocatenulatus lipase. [Thesis]

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Official URL: http://risc01.sabanciuniv.edu/record=b1534372 (Table of Contents)

Abstract

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 studies related to the protein engineering of BTL2 aiming to improve its chainlength selectivity and to assess the impact of the lid tryptophans. For both of these studies, the experimental approach was to generate specific BTL2 mutants for which site-directed mutagenesis, heterologous protein expression and purification methodologies were used.In the first study, a rational design of BTL2 was 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 was implemented to investigate the impacts of the two tryptophans (W212, W235) in the lid of BTL2. W235A affected tributyrin selectivity while W212A mutation is resulted in loss of the thermostability and aggregation tendency but also in the improvement of zinc tolerance in BTL2. For this, it is envisioned that the aggregation of BTL2 is mediated through a zinc domain and W212 is a secondary but vital residue for this action.

Item Type:Thesis
Uncontrolled Keywords:Protein engineering. -- Bacillus thermocatenulatus. -- Lipase. -- Selectivity. -- Stability. -- Protein mühendisliği. Lipaz. -- Seçicilik. -- Stabilite. -- Yönlendirilmiş moleküler dinamik.
Subjects:T Technology > TA Engineering (General). Civil engineering (General) > TA164 Bioengineering
ID Code:31238
Deposited By:IC-Cataloging
Deposited On:14 Apr 2017 15:16
Last Modified:14 Apr 2017 15:16

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