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.

Figure 1. Effect of whole-cell biocatalyst (WCB) pretreatment on ester synthesis activity.

Ten milligrams of each biocatalyst was used to convert 1% (w/w) water in tert-butanol over a period of 3 hours at 40°C. The control strain expressing the empty pET16b vector (open circles), the LipIAF5-2 strain without CTAB pretreatment (closed squares) and the strain with CTAB permeation (closed triangles) were employed. Results are averaged over three independent experiments.

Figure 2. Effect of temperature (A), water content (B), enzyme loading (C), and methanol molar ratio (D) on the LipIAF5-2 WCB activity.

A) Effect of temperature on the hydrolytic activity of the LipIAF5-2 WCB. Enzyme activities are expressed relative to maximal activity observed at 40°C (full squares). B) Effect of water content by percentage of substrate weight. Enzyme activities are expressed as final conversion of 1 mmol of glyceryl tributyrate and 4 mmol of methanol after 6 hours of incubation at 40°C. C) Effect of enzyme loading on the conversion of 1 mmol of glyceryl tributyrate using 4 molar equivalents of methanol as acyl acceptor in the aforementionned conditions. Conversion of glyceryl tributyrate (open circles) is expressed as final percentage of theoretical maximal yield (3 mmoles of product) after 3 hours of reaction. Initial specific activity (full squares) is expressed as the slope of liberated product determined under linear conditions over a period of at least 30 minutes. Biocatalyst amounts correspond to 1.65, 3.30, 6.61, 9.92 and 16.53% of substrate weight, respectively. D) Effect of alcohol molar ratios on the LipIAF5-2 WCB initial activity. Initial activity was determined in linear conditions over a 3-hour period using 10 mg of WCB, 5% water (w/w), 1 mmol of glyceryl tributyrate and various molar ratios (alcohol/substrate) of methanol in a final volume of 1.5 mL.

Figure 3. Conversion rates of LipIAF5-2 WCB transesterification and esterification toward acyl donors and acceptors.

A) The full bars represent the conversion of triglyceride substrates and the shaded gray bars the conversion of equivalent free fatty acids (FFA). The substrates employed are glyceryl triacetate and acetic acid (C2∶0), glyceryl tributyrate and tributyric acid (C4∶0), glyceryl trihexanoate and hexanoic acid (C6∶0) and glyceryl trioctanoate and octanoic acid (C8∶0). The results are expressed as the molar conversion of 1 mmol of each substrate after 6 hours of incubation at 40°C with 4 molar equivalents of methanol and 10 mg of LipIAF5-2 WCB. B) Impact of acyl acceptors on transesterification by LipIAF5-2 WCB. Transesterification of 1 mmol of glyceryl tributyrate was done using 4 molar equivalents of each primary alcohols for 3 hours at 40°C using 10 mg of LipIAF5-2 WCB and 10% (w/w) water. Results are averaged over two independent experiments performed in triplicate.

Figure 4. Time-course conversion of glyceryl triacetate into isoamyl acetate using the LipIAF5-2 WCB.

A) Reaction scheme for the transesterification of glyceryl triacetate into isoamyl acetate in presence of isoamyl alcohol. B) Time-course production of isoamyl acetate by LipIAF5-2 WCB immobilized in a fluidized bed reactor. C) Effect of LipIAF5-2 WCB recycling on the conversion yield of isoamyl acetate production. One gram of LipIAF5-2 WCB was loaded in an empty HPLC preparative column with a dead volume of 10 cm³ and placed in a heated chamber. The reaction mixture contained 50 mmoles of glyceryl triacetate as acyl donor and isoamyl alcohol at a molar ratio of 5∶1 with 5% water (w/w). The mixture was pumped in recirculating mode through the column at a flow rate of 1 mL/min for 24 h for each cycle. Cells were washed in open mode with 350 mL of tert-butanol between each cycle. All experiments were performed in triplicate.