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Abstract

An efficient mutagenesis strategy to create protein variants is the key to rapid evolution towards the desired properties of an enzyme.
The publication of the crystal structure of the Aspergillus niger epoxide hydrolase in 2000 and the discovery of the tunnel-shaped substrate binding site facilitated the application of a region-focused mutagenesis strategy. A strategy that placed the focus on the defined region (CAST) of the tunnel was used. Iterative CAST, instead of a simple single process, was carried out to mimic the Darwinian evolutionary process. This not only led to the accumulation of beneficial mutations, but also gave rise to possible cooperative effects. Furthermore, the results of this work also showed efficient and effective optimization of an enzyme at the genetic level. An overview of the results from iterative CASTing is shown inTable 5.
Combining this region-focused mutagenesis strategy, the activity selection system, and the MUX-ESI-MS screening method, a protein variant, LW202, with E-value greater than 100 was identified. This can be considered to be an excellent finding.^[82] Compared to the previous study of the same reaction using the same enzyme, in which an E-value of only 10.8 was attained employing the conventional approach epPCR,^[43] iterative CASTing proved to be more effective. Focus of later work has been placed on the characterization of this enzyme variant in order to gain further insights of its protein function and structure.