Biocatalysis, protein engineering, and flow chemistry are key enabling technologies that can yield drastically shorter and greener chemical processes. We sought to leverage these technologies to realize an improved synthesis. A cheap and green chemical commodity was identified as a starting material that allowed the generation of a key intermediate in the synthesis in a stepwise reaction. As an initial proof of concept, an enzyme-catalyzed reaction was demonstrated with modest yields and selectivity. However, the initial enzyme and process would not be sufficient to support large scale synthesis. To improve the synthesis, we engaged in enzyme evolution, enzyme immobilization, and flow chemistry. Through several rounds of evolution the enzyme was evolved to reach high conversion, selectivity and stability in the presence of organic solvents.

Learning Objectives:

1. Know protein engineering is often necessary to improve desired properties of an enzyme to be used in commercial manufacturing routes.

2. Recognize enzyme immobilization along with protein engineering can be effectively used to drive high product conversion in organic solvents without loosing enzymatic activity and without the need of enzymatic removal during reaction work up.