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      Short-Chain Flavor Ester Synthesis in Organic Media by an E. coli Whole-Cell Biocatalyst Expressing a Newly Characterized Heterologous Lipase

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          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.

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          Most cited references36

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          Improving enzymes by using them in organic solvents.

          The technological utility of enzymes can be enhanced greatly by using them in organic solvents rather than their natural aqueous reaction media. Studies over the past 15 years have revealed not only that this change in solvent is feasible, but also that in such seemingly hostile environments enzymes can catalyse reactions impossible in water, become more stable, and exhibit new behaviour such as 'molecular memory'. Of particular importance has been the discovery that enzymatic selectivity, including substrate, stereo-, regio- and chemoselectivity, can be markedly affected, and sometimes even inverted, by the solvent. Enzyme-catalysed reactions in organic solvents, and even in supercritical fluids and the gas phase, have found numerous potential applications, some of which are already commercialized.
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            Microbial lipases form versatile tools for biotechnology.

            K. Jaeger (1998)
            Lipases are secreted into the culture medium by many bacteria and fungi. They catalyse not only the hydrolysis but also the synthesis of long-chain acylglycerols. Important uses in biotechnology include their addition to detergents, the manufacture of food ingredients, pitch control in the pulp and paper industry, and biocatalysis of stereoselective transformations. This makes them the most widely used class of enzymes in organic chemistry. Immobilization in hydrophobic sol-gel matrices and in vitro evolution are promising novel approaches to increasing the stability or enantioselectivity, respectively, of lipases.
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              A review of the current state of biodiesel production using enzymatic transesterification.

              Enzymatic biodiesel production has been investigated intensively, but is presently employed industrially only in a 20,000 tons/year pilot plant in China (Du et al. [2008] Appl Microbiol Technol 79(3):331-337). This review presents a critical analysis of the current status of research in this area and accentuates the main obstacles to the widespread use of enzymes for commercial biodiesel transesterification. Improved results for enzymatic catalysis are seen with respect to increased yield, reaction time and stability, but the performance and price of the enzymes need further advances for them to become attractive industrially for biodiesel production. Critical aspects such as mass transfer limitations, use of solvents and water activity are discussed together with process considerations and evaluation of possible reactor configurations, if industrial production with enzymes is to be carried out. Results of published studies on the productivity of enzymes are also presented and compared to the use of chemical catalysts.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                26 March 2014
                : 9
                : 3
                : e91872
                Affiliations
                [1 ]INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada
                [2 ]Innu-Science Canada, Inc., Local 119, Trois-Rivières, Québec, Canada
                [3 ]PROTEO, the Québec Network for Research on Protein Function, Structure, and Engineering, Québec, Canada
                [4 ]GRASP, Groupe de Recherche Axé sur la Structure des Protéines, Québec, Canada
                Queen’s University Belfast, United Kingdom
                Author notes

                Competing Interests: YH was the research and development director of the commercial company Innu- Science Canada, Inc. GB’s salary was provided by a grant from the Canadian and Quebec Governments through the BMP Innovation scholarship program, aimed at fostering the development of university-industry partnerships. GB is a PhD student in FS’s and ND’s labs at INRS, and does not commercially benefit from the publication of this manuscript. As grant recipients from the National Sciences and Engineering Research Council of Canada, FS and ND are two academic professors who were involved in collaborative research projects with YH’s team at Innu-Science. FS and ND are not commercially involved with Innu-Science and neither owns stocks or shares in the company. YH and Innu-Science are fully committed to the full disclosure of all results and research materials resulting from the current research. All authors proclaim that this declaration of competing interests does not alter their adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: GB FS YH FL ND. Performed the experiments: GB FL. Analyzed the data: GB ND. Contributed reagents/materials/analysis tools: FS YH FL ND. Wrote the paper: GB ND.

                Article
                PONE-D-13-42215
                10.1371/journal.pone.0091872
                3966760
                24670408
                486f1b7c-3f83-420e-b962-10d6b1a22c1a
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 15 October 2013
                : 16 February 2014
                Page count
                Pages: 9
                Funding
                This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Collaborative Research and Development grant (awarded to FS) and NSERC Discovery grant RGPIN 402623-2011 (awarded to ND), as well as private funding from Innu-Science Canada. GB was the recipient of a BMP Innovation scholarship from NSERC and the Fonds de Recherche du Québec - Nature et Technologies. ND is also supported by a “Fonds de Recherche Québec – Santé” Research Scholar Junior 1 Career Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Biocatalysis
                Biotechnology
                Applied Microbiology
                Industrial Microbiology
                Environmental Biotechnology
                Small Molecules
                Microbiology
                Ecology and Environmental Sciences
                Environmental Protection

                Uncategorized
                Uncategorized

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