Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution.

ABSTRACT: Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorpora

SUBMITTER: Khersonsky O 

PROVIDER: S-EPMC3889864 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature