Biocatalysis in lipid modification, Greifswald 19–21 September 2012

Authors


Abstract

This three day event was organized by Uwe Bornscheuer and Pierre Villeneuve (division of biotechnology and enzyme technology of Euro Fed Lipid) in cooperation with SFEL (Société française pour l'étude des lipides) and the DGF (Deutsche Gesellschaft für Fettwissenschaft e.V.). The meeting was held at the Institute of Biochemistry of the Greifswald University, Greifswald – a small town at the Baltic coast of Mecklenburg-Vorpommern in northeastern Germany. About 60 scientists discussed recent developments in basic research and industrial process development involving enzymatic lipid modification. EJLST will publish a special issue based on the Greifswald meeting in late spring/early summer 2013; this meeting report will give you a flavor of what will be covered.

The meeting was opened on Wednesday Sep 19 with a keynote lecture given by I. Feussner (Göttingen, DE) on fatty acid double bond modifying enzymes in bacteria followed by a short session of speed talk poster presentations and a lively welcome reception.

On Thursday, the morning session started with two lectures by H. Brundiek (Greifswald, DE) and M. Adamczak (Olsztyn, PL) addressing the stability and selectivity of lipases. These enzymes are used for biotechnological purposes to improve the quality of products by selective modification of the respective substrates. The researchers made use of different specificities of lipases from Rhizopus, Candida, and Ustilago maydis. In his plenary lecture, F. Carrière (CNRS, Marseille, FR) addressed the role of another family of hydrolytic enzymes, the galactolipid hydrolyzing enzymes. Galactolipids, mainly monogalactosyl diglycerides (MGDG) and digalactosyl diglycerides (DGDG), are major classes of lipids found in plant membranes. Interestingly, the release of galactolipid fatty acids by galactolipases has not been intensively investigated. The focus of this lecture was on pancreatic lipase-related protein 2 (PLRP2), the main enzyme involved in the digestion of galactolipids in the gastrointestinal tract. Data on the biochemistry, the structure, and biotechnological application of this enzyme were presented.

Optimization of lipolytic reactions was the focus of the second session. E. Durand (Montpellier, FR) reported on the evaluation of deep eutectic solvents (DES) as new media for lipase-catalyzed reactions 1. DES result from the association of an ammonium salt and a hydrogen-bond donor. In these solvents, the hydrogen-bond donor interacts with the anion, inducing a depression in the melting point of the mixture. Advantages and limitations of several DES as “green solvents” for biotransformations with lipases were discussed. A. Aloulou (Sfax, TN) addressed the biochemical properties and potential uses of a Yarrowia lipolytica lipase in enzyme replacement therapy. Overexpressed lipases from various origins obtained by genetic engineering are actively required for the enzyme replacement therapy. Y. lipolytica lipase YLLIP2 is well suited for this purpose because it is not inhibited by bile salts and has a rather good stability at acidic pH. Furthermore, its regioselectivity towards triglycerides is comparable with that of pancreatic lipases. Finally, A. Marty (Toulouse, FR) presented a lecture dealing with the high potential of Yarrowia lipolytica for high-throughput screening of optimized enzymes, enzyme production, and lipid bioconversion or synthesis. Strains were developed and optimized for efficient lipase secretion and over-expression. A robotized process was optimized to obtain high reproducibility in order to minimize the level of false positives during enzyme evolution.

The afternoon session started with a presentation by P.M. Nielsen from Novozymes (Bagsvaerd, DK). Novozymes has been involved in the development of enzymatically catalyzed biodiesel production. In the presentation different enzymatic processes were discussed and the technical challenges and advantages outlined. Currently, the favored process is a transesterification with liquid formulated lipase followed by a caustic wash. G. Haraldsson (Reykjavik, IS) presented data concerning the synthesis of structured lipids controlled by highly regioselective lipases. The lecture was focused on chemoenzymatic production of various acylglycerol type lipids possessing pure EPA and DHA. This was accomplished by a two-step chemoenzymatic process with an immobilized Candida antarctica lipase (CAL). The last talk in this session was given by E. Dubreucq (Montpellier, FR) on applications of acyltransferases in lipid modifications. Enzymes with dual functions as lipase/acyltransferase, such as CpLIP2 from Candida parapsilosis preferentially catalyze acyltransfer reactions other than hydrolysis when a suitable nucleophile (alcohol, amine, thiol) is present in the reaction medium. This allows reactions such as alcoholysis, aminolysis, or perhydrolysis of lipids to be performed in aqueous media, where water is the solvent for the polar substrates and where lipid substrates and products constitute the organic phase. This method can be applied for various purposes such as fatty acid ethyl ester synthesis, fatty hydroxamic acid synthesis, fat interesterification, epoxidation, or acylation of peptides.

After a coffee and tea break, G. Daum (Graz, AT) addressed the life cycle of non-polar lipids in the baker's yeast Saccharomyces cerevisiae. He reported recent findings about storage and mobilization of TAG in this model microorganism. The multiple functions of the TAG lipases Tgl3p, Tgl4p, and Tgl5p as lipolytic enzymes but also as acyltransferases as well as regulatory aspects contributing to lipid homeostasis were discussed. P. Mugford (Dartmouth, CA) talked about lipase production of omega-3 concentrated triacylglycerides. Concentrated omega-3 fatty acids are commercially available in ethyl ester and triacylglyceride forms. Lipases can effectively convert these forms under mild conditions at an industrial scale. The limiting step in the production of enzymatic re-esterified triacylglycerides is the conversion of diacylglyceride to triacylglyceride. Finally, G. Lambeau (Valbonne, FR) introduced the family of secreted phospholipase A2. Originally identified in snake venoms, these enzymes were recently also detected in mammals. Individual sPLA2 enzymes exhibit unique tissue and cellular localizations, distinct gene regulation and highly specific enzymatic properties, suggesting various biological roles. They play a role in atherosclerosis, host defense, fertility, cancer, and membranous nephropathy, a human auto-immune kidney disease.

After the lectures, posters shown in this conference were presented in speed talks. In these 2-min presentations, young scientists pointed out highlights of their recent work. During the following poster session, the delegates had more time for discussions.

The day ended with a gorgeous conference dinner in the fisher village Wieck, which is located close to Greifswald. From the restaurant “Utkiek” meaning “Outlook” participants had indeed a great view over the shores of the Baltic Sea. During the dinner there was ample chance to continue discussing scientific problems but also to relax with a glass of beer or wine.

The morning session on Friday started with the keynote lecture by J. Ogawa (Kyoto, JP), who presented a deep analysis of the complex fatty acid saturation metabolism that allows gut bacteria such as Lactobacillus to detoxify PUFA by converting them into saturated fatty acids. The various types of molecular intermediates identified in these biohydrogenation pathways may be of great interest in various application sectors. Different examples of bioconversions using wild and engineered bacteria from gut were shown, allowing the production of a large range of rare fatty acids. F. Ergan (Laval, FR) presented recent work on the enrichment of algal phospholipid fractions with DHA using lipases active on phospholipids and able to discriminate between DHA and other fatty acids. This allowed obtaining phospholipid fractions containing up to 80% DHA. The optimization of biocatalytic continuous flow processes was discussed by R. de Souza (Rio de Janeiro, BR), comparing various lipase-catalyzed reactions in packed-bed and batch reactors to underline the importance of optimization methods in biocatalyzed process development 2.

Following the coffee break, questions related to the immobilization of lipases were further addressed by M. Hesseler (Llantrisant, UK), who presented strategies for the design and selection of immobilization supports for the development of optimized industrial processes. Different examples were used to show the impact of carrier properties and immobilization procedures on biocatalyst properties and process performances. I. Kurtovic (Nelson, NZ) showed how fish wastes could be a valuable source of enzymes. More specifically, applications of fish digestive lipases to the modification of flavor of dairy products were presented. These cheap and available enzymes are active at low temperatures and compare very well with commercial lipases and esterases in terms of flavor enhancement 3. The last morning talk by V. Recke (Braunschweig, DE) was dedicated to the synthesis of microbial glycolipid derivatives with modified bioactive and surface properties. The sugar part can be partly hydrolyzed using specific glycosidases (β-glucuronidase) and/or acylated using a lipase, yielding monoglycosylated and acylated derivatives with new properties.

After lunch, S. Ferreira-Dias (Lisbon, PT) discussed uses of new non-commercial lipases for the production of structured lipids. Results using naturally immobilized lipase from Carica papaya, immobilized lipase from Rh. orizae, and immobilized lipase/acyltransferase from C. parapsilosis were presented, showing different specificities and operational stabilities in bioreactors. A. Pinsolles (Bordeaux, FR) then presented the development of optimized reaction conditions for the synthesis of phosphatidylserine by transphosphatidylation of marine phospholipids with serine using phospholipase D as the biocatalyst. It was shown that the addition of bile salts to produce mixed micelles allowed the avoidance of organic solvents, which may be important for nutritional applications. The final talk was given by A. Steinbüchel (Münster, DE), who presented an overview of the diversity and the properties of bacterial acyltransferases, such as bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferases or 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylases. Acyltransferases are able to catalyze the synthesis of a wide range of esters in diverse metabolic pathways and offer a large potential for the synthesis of fine and bulk chemicals, such as tailor-made fatty acid esters, and polyhydroxyalcanoates.

After each lecture there was enough time for questions and discussions and with about 60 participants from 18 countries and 5 continents this symposium dedicated to biocatalysis in lipid modification was a great success. The majority of the delegates traveled from Greifswald directly to Poland to participate in the 10th Euro Fed Lipid Congress – see the Report on pages 252–254.

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