Zottig_2017_Protein.J_36_478

Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase alpha/beta hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 degrees C under neutral pH, but its maximal activity was detected at pH 10 and 45 degrees C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 degrees C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure-function relationships in lipases.