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.

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

“…Simply adding filter paper of appropriate cutoff to both ends of an empty HPLC column enables utilization of recombinant, lyophilized E. coli whole cell catalyst, as recently shown for the lipase-mediated conversion of glyceryl triacetate with methanol [73].…”

Enhancing the stability of a carboxylesterase by entrapment in chitosan coated alginate beads

“…The structure of the product was confirmed by NIST (National Institute of Standards and Technology) reference database ( Figure 7B). A short chain ester, isoamyl acetate (banana flavor), was synthesized with a recombinant whole cell biocatalyst of E. coli with various organic media and a good yield was obtained (Brault et al, 2014).…”

Lignocellulases: a review of emerging and developing enzymes, systems, and practices

“…The whole-cell systems convey several advantages such as stability, resistance, lower cost, reusability, and reduced labor, while providing products with high purity (Brault et al 2014;Kim et al 2014;Khattak et al 2014b). The reduced proneness to cell injury; improved resistance to physiological and environmental factors, such as variation in pH, elevated temperature, and system inhibition; high metabolic productivity; and reduced incubation time make the whole-cell system more promising for biotechnological implementation.…”