||Directed evolution: To examine the impact of circular permutation on catalysis, researchers
selected eight functional lipase B from Candida antarctica (CALB) variants with termini in or near
the active site for detailed kinetic characterization (Figure 2).
Following overexpression in P. pastoris, the proteins were purified
to homogeneity, and kinetic data for these variants were measured
on two standard lipase substrates, p-nitrophenol butyrate (pNB) and
6.8-difluoro-4-methylumbelliferyl (DiFMU) octanoate (table link).