- Production of flavours and fragrances via bioreduction of (4R)-(-)-carvone and (1R)-(-)-myrtenal by non-conventional yeast whole-cells
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As part of a program aiming at the selection of yeast strains which might be of interest as sources of natural flavours and fragrances, the bioreduction of (4R)-(-)-carvone and (1R)-(-)-myrtenal by whole-cells of non-conventional yeasts (NCYs) belonging to the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Kluyveromyces, Lindnera, Nakaseomyces, Vanderwaltozyma and Wickerhamomyces was studied. Volatiles produced were sampled by means of headspace solid-phase microextraction (SPME) and the compounds were analysed and identified by gas chromatography-mass spectroscopy (GC-MS). Yields (expressed as % of biotransformation) varied in dependence of the strain. The reduction of both (4R)-(-)-carvone and (1R)-(-)-myrtenal were catalyzed by some ene-reductases (ERs) and/or carbonyl reductases (CRs), which determined the formation of (1R,4R)-dihydrocarvone and (1R)-myrtenol respectively, as main flavouring products. The potential of NCYs as novel whole-cell biocatalysts for selective biotransformation of electron-poor alkenes for producing flavours and fragrances of industrial interest is discussed.
- Goretti, Marta,Turchetti, Benedetta,Cramarossa, Maria Rita,Forti, Luca,Buzzini, Pietro
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p. 5736 - 5748
(2013/07/19)
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- Molecular recognition in (+)-α-pinene oxidation by cytochrome P450cam
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Oxygenated derivatives of the monoterpene (+)-α-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-α-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450cam from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450cam for the selective oxidation of (+)-α-pinene. Consideration of the structures of (+)-camphor and (+)-α-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-α-pinene. Some mutants had tighter (+)-α-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-α-pinene oxidation rate of 270 nmol (nmol of P450cam)-1 min-1, which was 70% of the rate of camphor oxidation by wild-type P450cam. Camphor is oxidized by wild-type P450cam exclusively to 5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-α-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450cam enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-α-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450cam mutants initially examined, was determined to provide further insight into P450cam substrate binding and catalysis. (+)-α-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450cam mutant that could oxidize (+)-α-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-α-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450cam for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme - substrate interactions.
- Bell, Stephen G.,Chen, Xuehui,Sowden, Rebecca J.,Xu, Feng,Williams, Jennifer N.,Wong, Luet-Lok,Rao, Zihe
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p. 705 - 714
(2007/10/03)
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- Engineering the haem monooxygenase cytochrome P450cam for monoterpene oxidation
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Monooxygenated terpenes are fine fragrance and flavouring chemicals, and active site mutants of the haem monooxygenase cytochrome P450cam which were designed to have improved complementarity between the substrate binding pocket and the monoterp
- Bell,Sowden,Wong
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p. 635 - 636
(2007/10/03)
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- Pinane-Type Tridentate Reagents for Enantioselective Reactions: Reduction of Ketones and Addition of Diethylzinc to Aldehydes
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The reduction of aryl and alkenyl methyl ketones using lithium aluminum hydride modified with (1R,2S,3S,5A)-(+)-10-anilino-3-ethoxy-2-hydroxypinane (10b) afforded chiral secondary alcohols in 83-96% chemical yields and 50-91% ee with dominance of R enantiomers. The reduction of acetophenone in the presence of lithium iodide gave the alcohol product with higher ee. On the other hand, the addition reaction of diethylzinc to benzaldehyde using the pinane-based diols 5-9 as promoters gave 1-phenylpropanol in favor of the S enantiomer up to 88% ee. Using the pinane-based alcohols 10a-e as promoters, the R enantiomer was obtained as the major product. The addition reactions of diethylzinc to various substituted benzaldehydes, employing the diol ligands 5c and 8e, afforded predominantly the corresponding (S)-alcohols. The chiral modifiers 5-10 were prepared from (1R)-(-)-myrtenol and were readily recovered (>90%) after the asymmetric reactions. In this study, LAH reduction and Et2Zn addition are complementary methods for the preparation of optically active secondary alcohols. The ligand 10-butylanilino-2,3-dihydroxypinane 5c promoted the Et2Zn additions effectively, whereas the modifier 10-anilino-3-ethoxy-2-hydroxypinane 10b induced the LAH reductions in a highly enantioselective manner.
- Cherng, Yie-Jia,Fang, Jim-Min,Lu, Ta-Jung
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p. 3207 - 3212
(2007/10/03)
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- UNSOLVATED MAGNESIUM DIISOPROPYLAMIDE (MDA) IN ORGANIC SYNTHESIS. THE REDUCTION OF ALDEHYDES AND KETONES TO ALCOHOLS.
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A solution of unsolvated magnesium diisopropylamide in cyclohexane has been found to reduce aldehydes and ketones to the corresponding alcohols in good yield.
- Sanchez, Ramiro,Scott, William
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p. 139 - 142
(2007/10/02)
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