Chew Fajardo, Ying Lian
(2017).
Protein Engineering of the Calb Lipase to Synthesize Fragrance Compounds
Mémoire.
Québec, Université du Québec, Institut national de la recherche scientifique, maîtrise en microbiologie appliquée, 73 p.
Résumé
Abstract
A computationally-guided semi-rational protein design approach was used to improve
the enzymatic selectivity and catalytic efficiency of Pseudozyma antarctica lipase B
(CalB) to synthesize methyl salicylate and methyl cinnamate. These fatty acid esters
have significant relevance as flavoring and fragrance compounds in the biotechnological
industry. Moreover, CalB is a highly active lipase that is widely used for the enzymatic
hydrolysis and synthesis of esters, offering potential for the biological production of
flavoring agents. However, the relatively confined organization of its active site
precludes the recognition of bulky and aromatic substrates. To overcome this limitation,
in silico docking analyses of CalB were undertaken to identify amino acid residues
involved in precursor binding and recognition. These “hot spots” were subjected to
combinatorial mutagenesis to yield three generations of CalB libraries per substrate. A
surrogate substrate was used to screen for synthetic activity and evaluation of the new
CalB variants revealed mutations giving rise to significant increase in synthetic activity
relative to wild-type CalB. Ultimately, the best CalB variant could serve as a template to
develop an E. coli whole-cell biocatalyst suitable for industrial enzymatic synthesis of
methyl salicylate.
Type de document: |
Thèse
Mémoire
|
Directeur de mémoire/thèse: |
Doucet, Nicolas |
Mots-clés libres: |
CalB, semi-rational protein design, molecular docking, methyl salicylate,
directed evolution, protein engineering, lipase activity, enzyme catalysis. |
Centre: |
Centre INRS-Institut Armand Frappier |
Date de dépôt: |
02 sept. 2018 17:50 |
Dernière modification: |
02 sept. 2018 17:50 |
URI: |
http://espace.inrs.ca/id/eprint/7412 |
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