Protein Engineering of the Calb Lipase to Synthesize Fragrance Compounds

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.

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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:	https://espace.inrs.ca/id/eprint/7412

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