Thermoalkalophilic bacteria produce thermostable lipases that stood out among other lipases owing to their large potential for industrial processes operated at elevated temperatures. Identification of the factors enhancing thermostability is critical to efforts to design of promising biocatalysts. Hitherto a few studies have elucidated that the structural zinc and the conserved tryptophan that is closely located to structural zinc have potentiated thermostability of thermoalkalophilic lipases. In this study we investigate the impacts of the structural zinc and two tryptophans (W60, W211) that surround the zinc coordination site. For this project we chose the lipase from Bacillus thermocatenulatus (BTL2) as a representative of this family and delineated the interplay between zinc and the tryptophans in the context of thermostability by generating three mutants namely W60A, W211A and W60A/W211A. First of all the purity of our different samples was tested by SDS-Page in order to obtain the final list of trustable samples to be analysed along all the experiments. DLS was used to find which the sizes of the different types of lipases are. It shows us that there is not a remarkable different between BTL2 and the single mutants, whereas producing the double mutant means loosening of structure. The other alteration of structure to be proved was the removing of the zinc. The behavior of our proteins in a wide range of temperatures in function of their concentration and their zinc condition was tested performing thermostability assays. These illustrated how higher concentrations and zinc containing forms show more activity when the temperature is increased than the other forms. Thermal denaturation and CD experiments demonstrated that the zinc is essential for structural stability of BTL2 while the presence of tryptophans (W60, W211) has contributed to stability with the W211 being the most dominant. Overall these results imply that the conserved tryptophans have a direct impact on the zinc mediated thermostability of BTL2. In the meanwhile, structural investigations were conducted paying attention in the position of the tryptophans and the zinc positions. In the figures acquired we can see how the tryptophans protect the zinc cation.

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