Extended Data Fig. 4: Catalysed conversion of 10 and l-Ile to give 64.
From: Discovery, characterization and engineering of ligases for amide synthesis
a, Catalysed by PbCfaL (R395G/A294P) CLEAs. Reactions (100 mM Tris-HCl, 10 mM MgCl2, 10 mM ATP, 1 mM 10, 3 mM l-isoleucine, 50 ml total volume) were run for 24 h; the CLEA (cross-linked enzyme aggregate) was then removed, washed, and reintroduced to an identical reaction. While activity was seen to reduce over the 5 days, the CLEA still retained high levels of productivity even after 5 recycles over 5 days, whereas cell lysates generally precipitated and lost all activity within 12 h. Although CfaL undergoes extensive conformational changes during catalysis, encapsulating it within CLEAs shows the potential of immobilization to extend the functional lifespan of the CfaL. More sophisticated immobilization techniques may have the potential to further retain activity. Conversion values were calculated from HPLC peak area ratios of product and starting materials, and represent means where n = 5, error bars denote s.d. b, Catalysed by purified PbCfaL(R395G/A294P), showing percentage conversions of 10 and l-Ile in the presence of various solvents and at different concentrations. Conversion values were calculated from HPLC peak area ratios of product and starting materials and represent means where n = 3, error bars denote s.d.
