Different strategies to recover the activity of monomeric triosephosphate isomerase by directed evolution

A monomeric version of triosephosphate isomerase from Trypanosoma brucei, M onoTIM, has very low activity, and the same is true for all of the addition al monomeric variants so far constructed, Here, we subjected MonoTIM to dir ected evolution schemes to achieve an activity improvement. The constructio n of a suitable strain for genetic selection provided an effective way to o btain active catalysts from a diverse population of protein variants. We us ed this tool to identify active mutants from two different strategies of mu tagenesis: random mutagenesis of the whole gene and randomization of loop 2 . Both strategies converged in the isolation of mutations Ala43 to Pro and Thr44 to either Ala or Ser, when randomizing the entire gene or to Arg in t he case of randomization of loop 2. The kinetic characterization of the two more active mutants showed an increase of 11-fold in k(cat) and a reductio n of 4-fold in K-m for both of them, demonstrating the sensitivity of the s election method, A small difference in growth rate is observed when both mu tant genes are compared, which seems to be attributable to a difference in solubility of the expressed proteins.