title: Short-chain flavor ester synthesis in organic media by an E. coli whole-cell biocatalyst expressing a newly characterized heterologous lipase.
abstract: Short-chain aliphatic esters are small volatile molecules that produce fruity and pleasant aromas and flavors. Most of these esters are artificially produced or extracted from natural sources at high cost. It is, however, possible to 'naturally' produce these molecules using biocatalysts such as lipases and esterases. A gene coding for a newly uncovered lipase was isolated from a previous metagenomic study and cloned into E. coli BL21 (DE3) for overexpression using the pET16b plasmid. Using this recombinant strain as a whole-cell biocatalyst, short chain esters were efficiently synthesized by transesterification and esterification reactions in organic media. The recombinant lipase (LipIAF5-2) showed good affinity toward glyceryl trioctanoate and the highest conversion yields were obtained for the transesterification of glyceryl triacetate with methanol. Using a simple cetyl-trimethylammonium bromide pretreatment increased the synthetic activity by a six-fold factor and the whole-cell biocatalyst showed the highest activity at 40°C with a relatively high water content of 10% (w/w). The whole-cell biocatalyst showed excellent tolerance to alcohol and short-chain fatty acid denaturation. Substrate affinity was equally effective with all primary alcohols tested as acyl acceptors, with a slight preference for methanol. The best transesterification conversion of 50 mmol glyceryl triacetate into isoamyl acetate (banana fragrance) provided near 100% yield after 24 hours using 10% biocatalyst loading (w/w) in a fluidized bed reactor, allowing recycling of the biocatalyst up to five times. These results show promising potential for an industrial approach aimed at the biosynthesis of short-chain esters, namely for natural flavor and fragrance production in micro-aqueous media.
authors.affiliation: INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Brault
authors.affiliation: INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Shareck
authors.affiliation: Innu-Science Canada, Inc., Local 119, Trois-Rivières, Québec, Canada.
authors.forename: Yves
authors.initials: Y
authors.lastname: Hurtubise
authors.affiliation: INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Lépine
authors.affiliation: INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada; PROTEO, the Québec Network for Research on Protein Function, Structure, and Engineering, Québec, Canada; GRASP, Groupe de Recherche Axé sur la Structure des Protéines, Québec, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Doucet
mesh_terms: D061249:Batch Cell Culture Techniques; D055162:Biocatalysis / Q000187:drug effects*; D003470:Culture Media / Q000494:pharmacology*; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004951:Esterification / Q000187:drug effects; D004952:Esters / Q000378:metabolism*; D007700:Kinetics; D008049:Lipase / Q000302:isolation & purification / Q000378:metabolism*; D000432:Methanol / Q000737:chemistry; D009930:Organic Chemicals / Q000737:chemistry*; D000439:Pentanols / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012997:Solvents; D013649:Taste; D013696:Temperature; D013997:Time Factors; D014280:Triglycerides / Q000378:metabolism; D014867:Water / Q000737:chemistry
chemical_list: D003470:Culture Media; D004952:Esters; D009930:Organic Chemicals; D000439:Pentanols; D011994:Recombinant Proteins; D012997:Solvents; D014280:Triglycerides; D014867:Water; D008049:Lipase; D000432:Methanol; C020377:isoamyl acetate