| Summary: | Lipases are one of the most important biocatalysts for biotechnological applications. They have high importance since lipases have a catalytic activity that is related to their hydrolysis and synthesis reactions to high regioselectivity and enantioselectivity. They also have an activity over a wide range of temperature, pH, and diverse substrates. The aim of this master thesis project was to apply computational calculations to understand the reaction mechanism of the hydrolysis of p-nitrophenyl butyrate performed by the cold-adapted lipase, Bacillus pumilus Lipase A (BplA). Cold-adapted lipases are enzymes that have evolved to perform catalysis in a colder environment. The methods used for performing the computational calculations in this thesis project can be divided into three sections. The structure preparation, molecular dynamic (MD) simulations using NAMD, and quantum mechanics combined with molecular mechanics (QM/MM) using ORCA. Through eight substeps with MD and QM/MM implementations potential energy surfaces (PES), minimum energy path (MEP) and frequency calculations for the enzymatic reaction were provided. The rate-limiting step is the formation of the tetrahedral intermediate, the nucleophilic addition during the deacylation. The largest free energy barrier provided from the results had an activation free energy of 20.3 kcal/mol. The barriers of the deacylation were larger than the one for the acylation process. By understanding the reaction mechanism, one can understand how cold-adapted enzymes could catalyze the reaction to create lower energy barriers at low temperatures.
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