Improving the enantioselectivity of Bacillus subtilis LipA variants

Inácio, Vera

http://hdl.handle.net/10400.1/740

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Authors

Inácio, Vera

Advisor(s)

Quax, Wim

Costa, Mª Emília

Abstract(s)

This thesis describes the application of lipase A (lipA) from Bacillus subtilis 168 which is of particular importance for the production of enantiomer of interest S-(+)- 1, 2- O – isopropylidene-sn-glycerol (IPG) out of its racemic ester, as it shows some advantageous characteristics compared to other lipases. In directed evolution experiments, success strongly depends on the availability of screening or selection method. Enantioselectivity is a difficult parameter to select for. In a first approach, site-directed mutagenesis was employed to mutate the H156 from the catalytic triad of the enzyme and the mutants found had no activity as expected. In a second approach, directed evolution was used to evolve the lipase’s enantioselectivity and so the effect of the mutation of the inserted longer loop from Fusarium solani cutinase near the active site on both the activity and the enantioselectivity in an enzyme without a lid-like structure was assessed. The variants were enzymatically active with an inverted enantioselectivity towards S-(+)-IPG, however, the change in enantioselectivity was modest. This work, therefore, provides novel perspectives in the evolution of enzyme enantioselectivity. Directed evolution was applied to improve the enantioselectivity of the wild type enzyme to make it more suitable as (industrial) biocatalyst. To select for both enantioselectivity and catalytic activity of lipA, a growth selection system was used. An E. coli aspartate auxotrophic strain was transformed with a mutant library to select improved variants on minimal medium supplemented with an ester of enantiopure IPG coupled to aspartate. To impose selection pressure, a phosphonate suicide inhibitor coupled to the undesired enantiomer of IPG was added to the minimal medium as well. Thus, a dual selection system was used. Variants with an enantioselectivity towards S-(+)-IPG were selected. This system provides a powerfull tool in the selection of Bacillus lipA variants, in which both catalytic activity and enantioselectivity are taken into account. Moreover, this system is generally applicable in the selection of enantioselective hydrolases with any chiral substrate.