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. Author manuscript; available in PMC: 2020 Oct 4.
Published in final edited form as: Curr Opin Struct Biol. 2020 Jun 4;63:58–64. doi: 10.1016/j.sbi.2020.04.003

Figure 2. A practical two-step design strategy for enzyme optimization.

Figure 2

A stable and high-efficiency esterase was designed first by the PROSS stability-design method[33], which introduced 19 mutations in positions remote from the active site (red spheres) and then by the FuncLib function-enhancing method, which introduced four active-site mutations[43] (the active site is surface-colored in yellow and the FuncLib mutations are marked in blue spheres). This design exhibited higher stability, bacterial expression levels and approximately 1,000-fold higher esterase activity relative to the parental enzyme. Other enzymes reported in this study exhibited nearly 4,000-fold improvement in the hydrolysis of venomous nerve agents. This first stabilization step may address the stability-threshold challenge by improving the enzyme’s tolerance for function-enhancing mutations in the second step. For clarity, only one of the two chains comprising the enzyme is shown here.