Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE credits
The use of enzymes in the production of enantiomerically pure compounds is constantly increasing due to the many benefits and sustainabillity of the approach. Optically pure molecules are important building blocks in the pharmaceutical industry, as well as in other industrial biotechnological fields. The use of enzymes in kinetic resolution to obtain deracemization of these building blocks puts high demand on the stability, activity and selectivity of the catalysts. Enzyme engineering is commonly applied to improve these properties in order to reach a successful resolution system, and when structural information about an enzyme is available, it can be utilized to predict the outcome of certain point-mutations by molecular modeling.
The aim of this Master Thesis project was to improve the natural (∫)-selective nature of a serine protease denoted subtilisin Carlsberg towards secondary alcohols in a model transacylation reaction. To achieve this, a working expression system was set up. A his6-tag was added to the C-terminal of the enzyme gene construct, and the enzyme was expressed in Bacillus subtilis (B. subtilis). The wild type enzyme as well as all enzyme variants tested were immobilized on EziG™ beads and treated with two surfactants previously reported to have a positive impact on the selectivity of subtilisin Carlsberg in the literature.
The second tetrahedral intermediate of the reaction mechanism was built into an existing crystal structure using the YASARA software in order to identify suitable mutations, plausible to improve the selectivity. The assumption was made that the fit of methyl substituent of the alcohol substrate, in a medium-sized pocket of the active site, would have the greatest impact on the selectivity, and the mutants were selected based on this property alone. However, when the 7 enzyme variants selected and created was tested, it was clear that the fit in this pocket was not enough to explain the improved, unaltered or decreased selectivity of the variants. The results were evaluated further, focusing on steric hindrance between the residues surrounding the active site, and the phenyl group of the substrate, as well as the hydrogen bonds that are essential for the reaction mechanism to work. These results combined made it possible to draw valid conclustion about the obtained enantioselectivity of all variants.
In the end, two enzyme variants of improved enantioselectiviy was obtained; Met222Gly, and Met222Gly/Leu217Met. It was also shown that conducting the reaction in Tetrahydrofuran (THF) increased the selectivity, as well as the addition of the surfactants. However, the results need to be repeated and the variants should be tested using other substrates as well, but the results are promising.
enzyme, biocatalysis, protein engineering, subtilisin, Carlsberg, rational design