Structural and kinetic stability of the burkholderia glumae lipase

Stability is a crucial feature for enzymes employed in biocatalytic processes where they might be exposed to non-natural or even harsh environments. We have studied the behaviour of the lipase from Burkholderia glumae (BGL) by exposing it to challenging experimental conditions and characterizing changes in conformation and activity by a large set of biochemical, biophysical (mainly ESI-mass spectrometry, CD, fluorimetry) and computational methods. We observed that deactivation is not strictly related to structural instability in the assay conditions (temperature, pH, solvents), in fact (i) thermal deactivation precedes denaturation; (ii) acid-induced deactivation arises at higher pH than partial or global protein unfolding; and (iii) activity in most organic solvents decreases at solvent concentrations where conformation is fully retained. In the native protein, calcium is not accessible unless specific flexible loops are displaced, for example, by a temperature increase. Such movements concern the whole calcium-binding pocket and particularly the environment of the coordinating aspartate residue 241. As a consequence of metal depletion the protein unfolds irreversibly and undergoes aggregation. Our results are consistent with local unfolding phenomena causing deactivation and in a complex interplay between the mobility of loop structures and the ability of the protein to retain stabilizing Ca2+. This might suggest that a straightforward structural stabilization by site-directed mutagenesis aimed at increasing the general or local rigidity of the protein could be ineffective in relieving the subtle and elusive molecular effects that induce loss of activity in the “twilight” zone, i.e. under conditions that are mild enough to avoid protein unfolding but sub-optimal for activity. In this scenario, random approaches such as mutagenesis by directed evolution may prove to be more effective.