N-truncation in lipase Lip11 from Yarrowia lipolytica alleviates substrate inhibition with improved stability and efficiency ensuing distinct structural modifications

Four sequential N-truncations in the 70-residue extended terminus of lipase Lip11 yielded truncated variants T1 (Δ18), T2 (Δ35), T3 (Δ58), and T4 (Δ70). T1 and T2 instigated 35% reduction in activity, 20 U/mg to 12.5 U/mg; while N-truncating 58 residues generated 3.3-fold catalytically efficient variant T3 (Vmax = 43 U/mg), and further truncation (T4) emerged detrimental. Additionally, N-truncation abolished substrate inhibition in Lip11; and T3 was observed to form active aggregates at higher concentrations, migrating as dimers on SDS-PAGE. Further characterization of T3 revealed: higher thermal stability, 10 °C rise in temperature optima and half-life of >7 h at 50 °C; and enhanced acid-tolerance with 100% residual-activity at pH 5–7, and 80% at pH 3 and 4. Substrate specificity remained unaltered post-truncation as mid-chain specific with highest activity on castor oil (640 U/mg). Sequence variations were insufficient to confer thermal stability and structural modifications seemed plausible. Further far-UV CD analysis disclosed an increase in the β-sheet content from 8% to 16% upon N-truncation, possibly a structural basis for improved catalytic activity. Lastly, during tertiary structure analysis, T3 refolded exactly into its native conformation following thermal unfolding. And, it showed a higher surface hydrophobicity which correlated to improved thermal stability of T3.