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Characterization of a Novel Alkalophilic Lipase From Aneurinibacillus thermoaerophilus: Lid Heterogeneity and Assignment to Family I.5

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Abstract

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 α/β 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 °C under neutral pH, but its maximal activity was detected at pH 10 and 45 °C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 °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.

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Acknowledgements

X.Z. acknowledges a scholarship from CRMR (U. Laval, Canada). The editorial help of Pr. J. Turnbull in preparing this manuscript is acknowledged.

Funding

This work was supported by a NSERC Discovery grant awarded to M.B.

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Authors and Affiliations

  1. Département de chimie, biochimie et physique, Université du Québec Trois-Rivières, 3351 Boul. Des Forges, C.P. 500, Trois-Rivières, Québec, G9A 5H7, Canada

    Ximena Zottig, Fatma Meddeb-Mouelhi & Marc Beauregard

  2. PROTEO, Université Laval, 2705 Boul. Laurier, Québec, Québec, G1V 4G2, Canada

    Ximena Zottig, Fatma Meddeb-Mouelhi & Marc Beauregard

  3. Département de chimie, Université de Montréal, C.P. 6128 Succ. Centre-Ville, Montreal, Canada

    David M. Charbonneau

  4. Centre de recherche sur les matériaux lignocellulosiques, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Québec, G9A 5H7, Canada

    Marc Beauregard

Authors
  1. Ximena Zottig
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  2. Fatma Meddeb-Mouelhi
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  3. David M. Charbonneau
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  4. Marc Beauregard
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Contributions

XZ carried out all experimental work and wrote the initial draft. FM collaborated to production and purification protocols, and performed editorial work. DC collaborated to the in silico analyses and performed editorial work. MB supervised the research and wrote the final version of the manuscript.

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Correspondence to Marc Beauregard.

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The authors declare that they have no conflict of interest.

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Zottig, X., Meddeb-Mouelhi, F., Charbonneau, D.M. et al. Characterization of a Novel Alkalophilic Lipase From Aneurinibacillus thermoaerophilus: Lid Heterogeneity and Assignment to Family I.5. Protein J 36, 478–488 (2017). https://doi.org/10.1007/s10930-017-9743-9

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  • DOI: https://doi.org/10.1007/s10930-017-9743-9

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