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Worth to mention, there are no reports describing the application of isolated ene-reductases to reduce massoia lactone to δ-decalactone so far. This has prompted the investigation of reduction capability of isolated enzymes, such as OYE1 from Saccharomyces pastorianus and OYE2 and OYE3 obtained from S. Recently, ene-reductases have become available in sufficient quantities through the use of molecular biology techniques. However, this approach is limited by the difficulty of using high substrate concentrations and the necessity of long time of biotransformation. The production of δ-decalactone from massoia lactone by utilizing the reducing power of yeast ( Saccharomyces cerevisiae) and bacteria of the species Pseudomonas, Proteus and Bacillus have been reported in patents (EP0822259A1, US5763233) and in the literature 11, 12. These enzymes are already well known as catalysts allowing to obtain chiral building blocks for organic chemistry applications 8, 9, 10. The reduction of the endocyclic C=C double bond conjugated with the electron-withdrawing ester group can be catalysed by flavin-dependent ene-reductases which belong to the Old Yellow Enzyme (OYE) family (EC 1.6.99.1). One of the precursors of ( R)-(+)-δ-decalactone is ( R)-(–)-massoia lactone, which is commonly obtained by extraction from Cryptocaria massoia tree bark with high enantiomeric purity (ee > 99%) or prepared by synthesis 7. ( S)-(–)-δ-Decalactone odor description is peach, fatty, and buttery, whereas ( R)-(+)-δ-decalactone exhibits sweet, fruity and milky aroma properties and is used as additive in the production of cheese and butter 5. It is well-known that the odor quality of optically active substances is much superior than their racemic compounds. Many flavors and fragrances show optical activity.
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It is used to augment and enhance the aroma and taste of food preparations, chewing gums, beverages, toothpastes, colognes, perfumes and detergents 5, 6. δ-Decalactone, with its creamy, coconut-like, sweet, milky, fruit flavor, is of great interest to the industry. Their fragrance depends on the size of the ring, the type of substituents, the presence of unsaturated bonds, and the configuration of the stereogenic centers 3, 4. One group of additives with well-characterized flavor properties are compounds that contain ester bonds, including lactones, which are characterized by an intense, specific aroma, and which are widely used in the food, cosmetic, and pharmaceutical industries. Compounds obtained by biotransformation of natural starting materials are regarded as natural, according to United States and European Union regulations therefore, interest in the biotechnological production of natural flavor compounds has recently increased 2. An alternative route for flavor synthesis is based on application of enzymes or whole cells, which permits transformation with high chemo-, regio-, and stereoselectivity. It is due to low concentration of the desired compounds contained in raw materials 1. Extraction and isolation of natural food additives, especially flavors, is sometimes expensive. Increasing attention is being paid to the origin of food additives, and those with natural origin are preferred by customers.
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The process was intensified using a continuous-flow reactor-membrane filtration system (flow 0.1 mL/min, substrate concentration 10 mM, pH 7, 24 ☌) with cell lysate as biocatalyst combined with a cofactor regeneration system, which allowed obtaining > 99% bioconversion of massoia lactone. The biotransformation performed in batch mode, using Ca 2+-alginate immobilized cells of Escherichia coli BL21(DE3)/pET30a-OYE3, furnished the desired product with complete conversion in 30 min. The selected biocatalyst, either in the form of purified enzyme, cell lysate, whole cells or immobilized cells, was tested in the batch system as well as in the packed-bed flow bioreactor. Of 13 different ene-reductases isolated, purified and tested, OYE3 was found to be the most efficient biocatalyst. In this work, enantiomerically pure ( R)-(+)-δ-decalactone was obtained by reduction of the C=C double bond of natural massoia lactone in a continuous-flow reactor. Biotransformation has become an attractive approach to obtain natural products. The demand for natural food flavorings increases every year.