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Sample records for 2-3-pentanedione

  1. Effects of ammonium lactate on 2,3-pentanedione formation from lactic acid

    Tam, M.S.; Jackson, J.E.; Miller, D.J.

    1999-10-01

    Ammonia is often used for pH adjustment during fermentation of glucose to lactic acid. Its presence as ammonium lactate in the catalytic upgrading of lactic acid to 2,3-pentanedione over CsOH/silica reduces diketone yields to nearly zero at high ammonia levels. Removal of ammonia from the feed restores 2,3-pentanedione yield, indicating that the catalyst itself is not poisoned by ammonia. Instead, 2,3-pentanedione continues to form in the presence of ammonia and is consumed in secondary reactions downstream of the catalyst bed. Both base-catalyzed self-condensation of 2,3-pentanedione to duroquinone (and oligomeric species) and direct reaction of ammonia with the diketone are observed.

  2. Atmospheric chemistry of 2,3-pentanedione: photolysis and reaction with OH radicals.

    Szabó, Emese; Djehiche, Mokhtar; Riva, Matthieu; Fittschen, Christa; Coddeville, Patrice; Sarzyński, Dariusz; Tomas, Alexandre; Dóbé, Sándor

    2011-08-25

    The kinetics of the overall reaction between OH radicals and 2,3-pentanedione (1) were studied using both direct and relative kinetic methods at laboratory temperature. The low pressure fast discharge flow experiments coupled with resonance fluorescence detection of OH provided the direct rate coefficient of (2.25 ± 0.44) × 10(-12) cm(3) molecule(-1) s(-1). The relative-rate experiments were carried out both in a collapsible Teflon chamber and a Pyrex reactor in two laboratories using different reference reactions to provide the rate coefficients of 1.95 ± 0.27, 1.95 ± 0.34, and 2.06 ± 0.34, all given in 10(-12) cm(3) molecule(-1) s(-1). The recommended value is the nonweighted average of the four determinations: k(1) (300 K) = (2.09 ± 0.38) × 10(-12) cm(3) molecule(-1) s(-1), given with 2σ accuracy. Absorption cross sections for 2,3-pentanedione were determined: the spectrum is characterized by two wide absorption bands between 220 and 450 nm. Pulsed laser photolysis at 351 nm was used and the depletion of 2,3-pentanedione (2) was measured by GC to determine the photolysis quantum yield of Φ(2) = 0.11 ± 0.02(2σ) at 300 K and 1000 mbar synthetic air. An upper limit was estimated for the effective quantum yield of 2,3-pentanedione applying fluorescent lamps with peak wavelength of 312 nm. Relationships between molecular structure and OH reactivity, as well as the atmospheric fate of 2,3-pentanedione, have been discussed.

  3. Determina tion of diacetyl and 2,3-pentanedione in beer by gc/ms using solid-phase extraction columns

    Pejin Jelena D.

    2002-01-01

    Full Text Available A new GC/MS method for the determination of diacetyl and 2,3-pentanedione was investigated. Diacetyl and 2,3-pentanedione were derivatized with 1,2-diaminobenzene to form 2,3-dimetylquinoxaline and 2-ethyl-3-methylquinoxaline. respectively. The amounts of formed 2.3-dimetylqu:inoxaline and 2-ethyl-3,.methylquinoxaline were proportional to the concentrations of diacetyl and 2,3-penianedione present in the sample. 2,3-Dimetylquinoxaline and 2-ethyl-3-methylquinoxaline were extracted by solid-phase extraction (SPE columns and determined by gas chromatography using a mass selective detector. This method was applied for the determination of diacetyl and 2,3-pentanedione concentrations in beer. Extraction by SPE columns proved to be very simple and reliable. The method can be used for simultaneous determination of diacetyl and 2,3-pentanedione concentrations in beer in a great number of beer samples.

  4. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

    Zaccone, Eric J.; Goldsmith, W. Travis; Shimko, Michael J.; Wells, J.R.; Schwegler-Berry, Diane; Willard, Patsy A.; Case, Shannon L.; Thompson, Janet A.; Fedan, Jeffrey S.

    2016-01-01

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport, without affecting Cl− transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. PMID:26454031

  5. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents.

    Zaccone, Eric J; Goldsmith, W Travis; Shimko, Michael J; Wells, J R; Schwegler-Berry, Diane; Willard, Patsy A; Case, Shannon L; Thompson, Janet A; Fedan, Jeffrey S

    2015-12-15

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance,we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity.We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport,without affecting Cl- transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100-360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro.

  6. Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewer's wort.

    Krogerus, Kristoffer; Gibson, Brian R

    2013-08-01

    Undesirable butter-tasting vicinal diketones are produced as by-products of valine and isoleucine biosynthesis during wort fermentation. One promising method of decreasing diacetyl production is through control of wort valine content since valine is involved in feedback inhibition of enzymes controlling the formation of diacetyl precursors. Here, the influence of valine supplementation, wort amino acid profile and free amino nitrogen content on diacetyl formation during wort fermentation with the lager yeast Saccharomyces pastorianus was investigated. Valine supplementation (100 to 300 mg L(-1)) resulted in decreased maximum diacetyl concentrations (up to 37 % lower) and diacetyl concentrations at the end of fermentation (up to 33 % lower) in all trials. Composition of the amino acid spectrum of the wort also had an impact on diacetyl and 2,3-pentanedione production during fermentation. No direct correlation between the wort amino acid concentrations and diacetyl production was found, but rather a negative correlation between the uptake rate of valine (and also other branched-chain amino acids) and diacetyl production. Fermentation performance and yeast growth were unaffected by supplementations. Amino acid addition had a minor effect on higher alcohol and ester composition, suggesting that high levels of supplementation could affect the flavour profile of the beer. Modifying amino acid profile of wort, especially with respect to valine and the other branched-chain amino acids, may be an effective way of decreasing the amount of diacetyl formed during fermentation.

  7. Flavoring Chemicals in E-Cigarettes: Diacetyl, 2,3-Pentanedione, and Acetoin in a Sample of 51 Products, Including Fruit-, Candy-, and Cocktail-Flavored E-Cigarettes

    Allen, Joseph G.; Flanigan, Skye S.; LeBlanc, Mallory; Vallarino, Jose; MacNaughton, Piers; Stewart, James H.; Christiani, David C.

    2015-01-01

    Background: There are > 7,000 e-cigarette flavors currently marketed. Flavoring chemicals gained notoriety in the early 2000s when inhalation exposure of the flavoring chemical diacetyl was found to be associated with a disease that became known as “popcorn lung.” There has been limited research on flavoring chemicals in e-cigarettes. Objective: We aimed to determine if the flavoring chemical diacetyl and two other high-priority flavoring chemicals, 2,3-pentanedione and acetoin, are present in a convenience sample of flavored e-cigarettes. Methods: We selected 51 types of flavored e-cigarettes sold by leading e-cigarette brands and flavors we deemed were appealing to youth. E-cigarette contents were fully discharged and the air stream was captured and analyzed for total mass of diacetyl, 2,3-pentanedione, and acetoin, according to OSHA method 1012. Results: At least one flavoring chemical was detected in 47 of 51 unique flavors tested. Diacetyl was detected above the laboratory limit of detection in 39 of the 51 flavors tested, ranging from below the limit of quantification to 239 μg/e-cigarette. 2,3-Pentanedione and acetoin were detected in 23 and 46 of the 51 flavors tested at concentrations up to 64 and 529 μg/e-cigarette, respectively. Conclusion: Because of the associations between diacetyl and bronchiolitis obliterans and other severe respiratory diseases observed in workers, urgent action is recommended to further evaluate this potentially widespread exposure via flavored e-cigarettes. Citation: Allen JG, Flanigan SS, LeBlanc M, Vallarino J, MacNaughton P, Stewart JH, Christiani DC. 2016. Flavoring chemicals in e-cigarettes: diacetyl, 2,3-pentanedione, and acetoin in a sample of 51 products, including fruit-, candy-, and cocktail-flavored e-cigarettes. Environ Health Perspect 124:733–739; http://dx.doi.org/10.1289/ehp.1510185 PMID:26642857

  8. 76 FR 1434 - Request for Information on 2,3-Pentanedione and Other Alpha-Diketones Used As Diacetyl Substitutes

    2011-01-10

    ...: (1) Published and unpublished reports and findings from in vitro and in vivo toxicity studies with 2... other alpha-diketones; (6) Research findings from in vitro and in vivo toxicity studies; (7)...

  9. Environmental characterization of a coffee processing workplace with obliterative bronchiolitis in former workers.

    Duling, Matthew G; LeBouf, Ryan F; Cox-Ganser, Jean M; Kreiss, Kathleen; Martin, Stephen B; Bailey, Rachel L

    2016-10-02

    Obliterative bronchiolitis in five former coffee processing employees at a single workplace prompted an exposure study of current workers. Exposure characterization was performed by observing processes, assessing the ventilation system and pressure relationships, analyzing headspace of flavoring samples, and collecting and analyzing personal breathing zone and area air samples for diacetyl and 2,3-pentanedione vapors and total inhalable dust by work area and job title. Mean airborne concentrations were calculated using the minimum variance unbiased estimator of the arithmetic mean. Workers in the grinding/packaging area for unflavored coffee had the highest mean diacetyl exposures, with personal concentrations averaging 93 parts per billion (ppb). This area was under positive pressure with respect to flavored coffee production (mean personal diacetyl levels of 80 ppb). The 2,3-pentanedione exposures were highest in the flavoring room with mean personal exposures of 122 ppb, followed by exposures in the unflavored coffee grinding/packaging area (53 ppb). Peak 15-min airborne concentrations of 14,300 ppb diacetyl and 13,800 ppb 2,3-pentanedione were measured at a small open hatch in the lid of a hopper containing ground unflavored coffee on the mezzanine over the grinding/packaging area. Three out of the four bulk coffee flavorings tested had at least a factor of two higher 2,3-pentanedione than diacetyl headspace measurements. At a coffee processing facility producing both unflavored and flavored coffee, we found the grinding and packaging of unflavored coffee generate simultaneous exposures to diacetyl and 2,3-pentanedione that were well in excess of the NIOSH proposed RELs and similar in magnitude to those in the areas using a flavoring substitute for diacetyl. These findings require physicians to be alert for obliterative bronchiolitis and employers, government, and public health consultants to assess the similarities and differences across the industry to

  10. Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation

    Zhu, Jie; Hixon, Mark S.; Globisch, Daniel; Kaufmann, Gunnar F.; Janda, Kim D.

    2013-01-01

    In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2 related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet t...

  11. Evaluation of the hypersensitivity potential of alternative butter flavorings.

    Anderson, Stacey E; Franko, Jennifer; Wells, J R; Lukomska, Ewa; Meade, B Jean

    2013-12-01

    Concern has been raised over the association of diacetyl with lung disease clinically resembling bronchiolitis obliterans in food manufacturing workers. This has resulted in the need for identification of alternative chemicals to be used in the manufacturing process. Structurally similar chemicals, 2,3-pentanedione, 2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione, used as constituents of synthetic flavoring agents have been suggested as potential alternatives for diacetyl, however, immunotoxicity data on these chemicals are limited. The present study evaluated the dermal irritation and sensitization potential of diacetyl alternatives using a murine model. None of the chemicals were identified as dermal irritants when tested at concentrations up to 50%. Similar to diacetyl (EC3=17.9%), concentration-dependent increases in lymphocyte proliferation were observed following exposure to all four chemicals, with calculated EC3 values of 15.4% (2,3-pentanedione), 18.2% (2,3-hexanedione), 15.5% (3,4-hexanedione) and 14.1% (2,3-heptanedione). No biologically significant elevations in local or total serum IgE were identified after exposure to 25-50% concentrations of these chemicals. These results demonstrate the potential for development of hypersensitivity responses to these proposed alternative butter flavorings and raise concern about the use of structurally similar replacement chemicals. Additionally, a contaminant with strong sensitization potential was found in varying concentrations in diacetyl obtained from different producers.

  12. Identification of predominant aroma components of raw, dry roasted and oil roasted almonds.

    Erten, Edibe S; Cadwallader, Keith R

    2017-02-15

    Volatile components of raw, dry roasted and oil roasted almonds were isolated by solvent extraction/solvent-assisted flavor evaporation and predominant aroma compounds identified by gas chromatography-olfactometry (GCO) and aroma extract dilutions analysis (AEDA). Selected odorants were quantitated by GC-mass spectrometry and odor-activity values (OAVs) determined. Results of AEDA indicated that 1-octen-3-one and acetic acid were important aroma compounds in raw almonds. Those predominant in dry roasted almonds were methional, 2- and 3-methylbutanal, 2-acetyl-1-pyrroline and 2,3-pentanedione; whereas, in oil roasted almonds 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 2,3-pentanedione, methional and 2-acetyl-1-pyrroline were the predominant aroma compounds. Overall, oil roasted almonds contained a greater number and higher abundance of aroma compounds than either raw or dry roasted almonds. The results of this study demonstrate the importance of lipid-derived volatile compounds in raw almond aroma. Meanwhile, in dry and oil roasted almonds, the predominant aroma compounds were derived via the Maillard reaction, lipid degradation/oxidation and sugar degradation.

  13. The involvement of bacterial quorum sensing in the spoilage of refrigerated Litopenaeus vannamei.

    Zhu, Suqin; Wu, Haohao; Zeng, Mingyong; Liu, Zunying; Wang, Ying

    2015-01-02

    Quorum-sensing signals in refrigerated shrimp (Litopenaeus vannamei) undergoing spoilage were examined using bioreporter assays, thin-layer chromatography and gas chromatography-mass spectrometry, and the results revealed the presence of three types of autoinducers including acetylated homoserine lactones (AHLs) (i.e., N-hexanoyl-homoserine lactone, N-oxohexanoyl-homoserine lactone and N-octanoyl-homoserine lactone), autoinducer-2, and cyclic dipeptides (i.e., cyclo-(L-Pro-L-Leu), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe)). Autoinducer-2, rather than any AHL, was detected in extracts from pure cultures of the specific spoilage organisms (SSO), i.e., Shewanella putrefaciens (SS01) and Shewanella baltica (SA02). As for the cyclic peptides, only SA02 was determined to produce cyclo-(L-Pro-L-Leu). According to the transcription levels of LuxR (the master quorum-sensing regulator) in the SSO in response to exogenous autoinducers, the SSO could sense AHLs and cyclo-(L-Leu-L-Leu), rather than autoinducer-2, cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe). In accordance with the results of LuxR expression, the production of biofilm matrixes and extracellular proteases in the SSO was regulated by exogenous AHLs and cyclo-(L-Pro-L-Leu), rather than 4,5-dihydroxy-2,3-pentanedione (the autoinducer-2 precursor), cyclo-(L-Leu-L-Leu) and cyclo-(L-Pro-L-Phe). Exogenous N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) increased the growth rates and population percentages of the SSO in shrimp samples under refrigerated storage, and interestingly, exogenous 4,5-dihydroxy-2,3-pentanedione also increased the population percentages of the SSO in vivo by inhibiting the growth of the competing bacteria. However, according to the levels of TVB-N and the volatile organic components in the shrimp samples, exogenous 4,5-dihydroxy-2,3-pentanedione did not accelerate the shrimp spoilage process as N-hexanoyl-homoserine lactone and cyclo-(L-Pro-L-Leu) did. In summary, our results suggest that

  14. Optimal design of experiments applied to headspace solid phase microextraction for the quantification of vicinal diketones in beer through gas chromatography-mass spectrometric detection.

    Leça, João M; Pereira, Ana C; Vieira, Ana C; Reis, Marco S; Marques, José C

    2015-08-05

    Vicinal diketones, namely diacetyl (DC) and pentanedione (PN), are compounds naturally found in beer that play a key role in the definition of its aroma. In lager beer, they are responsible for off-flavors (buttery flavor) and therefore their presence and quantification is of paramount importance to beer producers. Aiming at developing an accurate quantitative monitoring scheme to follow these off-flavor compounds during beer production and in the final product, the head space solid-phase microextraction (HS-SPME) analytical procedure was tuned through experiments planned in an optimal way and the final settings were fully validated. Optimal design of experiments (O-DOE) is a computational, statistically-oriented approach for designing experiences that are most informative according to a well-defined criterion. This methodology was applied for HS-SPME optimization, leading to the following optimal extraction conditions for the quantification of VDK: use a CAR/PDMS fiber, 5 ml of samples in 20 ml vial, 5 min of pre-incubation time followed by 25 min of extraction at 30 °C, with agitation. The validation of the final analytical methodology was performed using a matrix-matched calibration, in order to minimize matrix effects. The following key features were obtained: linearity (R(2) > 0.999, both for diacetyl and 2,3-pentanedione), high sensitivity (LOD of 0.92 μg L(-1) and 2.80 μg L(-1), and LOQ of 3.30 μg L(-1) and 10.01 μg L(-1), for diacetyl and 2,3-pentanedione, respectively), recoveries of approximately 100% and suitable precision (repeatability and reproducibility lower than 3% and 7.5%, respectively). The applicability of the methodology was fully confirmed through an independent analysis of several beer samples, with analyte concentrations ranging from 4 to 200 g L(-1).

  15. 分散液相微萃取/高效液相色谱法同时测定啤酒中3种酮类老化物质%Simultaneous Determination of Three Keto Ageing Compounds in Beer by DLLME/HPLC

    郭静; 徐烨; 李琦; 王乃芝; 刘丽华

    2012-01-01

    A method for the simultaneous determination of three main keto ageing compounds, such as diacetyl, 2, 3-pentanedione and β-damascone, in beer by dispersive liquid — liquid microextrac-tion/high performance liquid chromatography was proposed. Derivatization conditions of diacetyl and 2, 3-pentanedione and high performance liquid chromatography conditions of the three target components were established. The separation and detection of three keto ageing compounds were performed in 15 min. The conditions of dispersive liquid -liquid microextraction were studied, and the enrichment factors of three compounds were 45, 63 and 121, respectively. Good linearities were obtained for three compounds with correlation coefficients of 0. 999 6-0.999 9, and the linear range is greater than or equal to 103. The detection limits ( S/N = 3 ) were in the range of 0. 3 -0. 9 μg/L. The average recoveries of real samples were in the range of 86% -97% with RSDs of 2.4% -4. 8% .%建立了分散液相微萃取(DLLME)/高效液相色谱(HPLC)同时测定啤酒中双乙酰、2,3-戊二酮和β-大马酮3种主要酮类老化物质的方法.确定了双乙酰和2,3-戊二酮的衍生化反应条件和3种目标组分同时检测的高效液相色谱条件,在15 min内实现了对3种酮类老化物质的分离和检测;优化了分徽液相徽萃取的实验条件,使3种目标组分得到同时提取和富集,各组分的富集倍数分别为45、63、121倍.所建立的3种组分的工作曲线相关系数良好(r为0.9996~0.9999),线性范围可达3~4个数量级,检出限(S/N=3)为0.3~0.9 μg/L,样品的加标回收率为86%~97%,相对标准偏差为2.4% ~4.8%.

  16. The response of Serratia marcescens JG to environmental changes by quorum sensing system.

    Sun, Shu-Jing; Liu, Hui-Jun; Weng, Cai-Hong; Lai, Chun-Fen; Ai, Liu-Ying; Liu, Yu-Chen; Zhu, Hu

    2016-08-01

    Many bacterial cells are known to regulate their cooperative behaviors and physiological processes through a molecular mechanism called quorum sensing. Quorum sensing in Serratia marcescens JG is mediated by the synthesis of autoinducer 2 (AI-2) which is a furanosyl borate diester. In this study, the response of quorum sensing in S. marcescens JG to environment changes such as the initial pH, carbon sources and boracic acid was investigated by a bioreporter and real-time PCR analysis. The results show that glucose can affect AI-2 synthesis to the greatest extent, and 2.0 % glucose can stimulate S. marcescens JG to produce more AI-2, with a 3.5-fold increase in activity compared with control culture. Furthermore, the response of quorum sensing to changes in glucose concentration was performed by changing the amount of luxS RNA transcripts. A maximum of luxS transcription appeared during the exponential growth phase when the glucose concentration was 20.0 g/L. AI-2 production was also slightly impacted by the low initial pH. It is significant for us that the addition of boracic acid at microdosage (0.1-0.2 g/L) can also induce AI-2 synthesis, which probably demonstrated the feasible fact that the 4,5-dihydroxy-2, 3-pentanedione cyclizes by the addition of borate and the loss of water, is hydrated and is converted to the final AI-2 in S. marcescens JG.

  17. Probing the Catalytic Mechanism of S-Ribosylhomocysteinase (LuxS) with Catalytic Intermediates and Substrate Analogues

    Gopishetty, Bhaskar; Zhu, Jinge; Rajan, Rakhi; Sobczak, Adam J.; Wnuk, Stanislaw F.; Bell, Charles E.; Pei, Dehua; (OSU); (FIU)

    2009-05-12

    S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). The catalytic mechanism of LuxS comprises three distinct reaction steps. The first step involves carbonyl migration from the C1 carbon of ribose to C2 and the formation of a 2-ketone intermediate. The second step shifts the C=O group from the C2 to C3 position to produce a 3-ketone intermediate. In the final step, the 3-ketone intermediate undergoes a {beta}-elimination reaction resulting in the cleavage of the thioether bond. In this work, the 3-ketone intermediate was chemically synthesized and shown to be chemically and kinetically competent in the LuxS catalytic pathway. Substrate analogues halogenated at the C3 position of ribose were synthesized and reacted as time-dependent inhibitors of LuxS. The time dependence was caused by enzyme-catalyzed elimination of halide ions. Examination of the kinetics of halide release and decay of the 3-ketone intermediate catalyzed by wild-type and mutant LuxS enzymes revealed that Cys-84 is the general base responsible for proton abstraction in the three reaction steps, whereas Glu-57 likely facilitates substrate binding and proton transfer during catalysis.

  18. Autoinducer 2: a concentration-dependent signal for mutualistic bacterial biofilm growth

    Rickard, A.H.; Palmer, R.J.; Blehert, D.S.; Campagna, S.R.; Semmelhack, M.F.; Egland, P.G.; Bassler, B.L.; Kolenbrander, P.E.

    2006-01-01

    4,5-dihydroxy-2,3-pentanedione (DPD), a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). AI-2 is proposed to be a universal signal molecule mediating interspecies communication among bacteria. We show that mutualistic and abundant biofilm growth in flowing saliva of two human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by genetic or chemical (synthetic AI-2 in the form of DPD) complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Furthermore, an optimal concentration of DPD was determined, above and below which biofilm formation was suppressed. The optimal concentration was 100-fold lower than the detection limit of the currently accepted AI-2 assay. Thus, AI-2 acts as an interspecies signal and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity. ?? 2006 Blackwell Publishing Ltd.

  19. Volatile compounds of some popular Mediterranean seafood species

    I. GIOGIOS

    2013-07-01

    Full Text Available The volatile compounds of highly commercialised fresh Mediterranean seafood species, including seven fish (sand-smelt Atherina boyeri, picarel Spicara smaris, hake Merluccius merluccius, pilchard Sardina pilchardus, bogue Boobps boops, anchovy Engraulis encrasicolus and striped-mullet Mullus barbatus, squid (Loligo vulgaris, shrimp (Parapenaeus longirostris and mussel (Mytilus galloprovincialis, were evaluated by simultaneous steam distillation-extraction and subsequent GC-MS analysis. A total of 298 volatile compounds were detected. The mussels contained the highest total concentration of volatile compounds, while pilchard among fish species contained the highest number and concentrations of volatile compounds. Individual patterns of volatile compounds have been distinguished. The fish species when compared to the shellfish species studied, contained 6 to 30 times more 1-penten-3-ol, higher quantities of 2-ethylfuran, and 2,3-pentanedione, which was absent from the shellfish species. Pilchard is characterized by a high concentration of alcohols, shrimps by the high presence of amines and S-compounds, while mussels by high amounts of aldehydes, furans, and N-containing compounds (pyridine, pyrazines and pyrrols. The fatty acid-originating carbonyl compounds in fish seem to be related to the species’ fat content.

  20. Odor-Active (E -4-Methyl-3-hexenoic Acid in Roasted Coffee Generated in the Maillard Reactions of L-Isoleucine with Sugars

    Hironari Miyazato

    2013-10-01

    Full Text Available This study describes a novel odor-active unsaturated aliphatic acid in roasted Brazilian Arabica coffee. (E-4-Methyl-3-hexenoic acid -4M3H, which is responsible for a sweaty odor, was identified for the first time using Gas Chromatography-Mass Spectrometry/Olfactometry (GC-MS/O, Aroma Extract Dilution Analysis (AEDA and Multidimensional Gas Chromatography-Mass Spectrometry (MDGC-MS; its (Z -isomer was also determined. Additionally, we conducted a model formation experiment to detect the presence of 4M3H in coffee and found that it may be produced nonstereoselectively in the Maillard reactions of L-isoleucine with sugars (xylose, fructose, glucose, rhamnose and sucrose. We also found that 2-methylbutanal derived from L-isoleucine and sugar degradation compounds such as α-dicarbonyl compounds (glyoxal, 2-oxopropanal, 2, 3-butanedione and 2, 3-pentanedione and α-hydroxy ketones (1-hydroxy-2-propanone and 3-hydroxy-2-butanone are key factors in the formation of 4M3H. Based on these results, we propose a series of potential nonstereoselective formation pathways for 4M3H.

  1. IDENTIFIKASI CHARACTER IMPACT ODORANTS BUAH KAWISTA (Feronia Limonia [Identification of Character Impact Odorants of Wood Apple Fruit (Feronia Limonia

    Anton Apriyantono1

    2004-04-01

    Full Text Available The volatiles of the kawista fruit (wood apple were analyzed by gas chromatography (GC and a combined gas chromatography-mass spectrometer (GC-MS. Character impact odorants of the fruits were systematically characterized by aroma extract dilution analysis (AEDA with GC-Olfactometry (GC-O. A total of 75 compounds were identified, including 28 esters, 11 alcohols, 10 aldehydes, 1 acetal, 10 ketones, 4 lactones, 1 heterocyclic, 4 aliphatic hydrocarbons, 1 furan and 5 acids. However, only 44 volatiles were detected by GC-O. Among these, compounds with the most impact were ethyl butyrate (fruity, sweet, banan-likeand methyl butyrate (fruit, sour with a flavor dilution factor of 256 and 64, respectively. Based on AEDA results, butyric acid, 3-mathyl valeric acid, 1-octen-3-ol, pentyl isobutyrate, 2-ethyl hexanoic acid, ethyl octanoate, gamma-decalactone, 2,3-pentanedione, 3-octanone, 5-methyl-3-heptanone, 9-methyl-5-undecene and (E-2-hexenyl butyrate seem to contribute to kawista fruit flavor

  2. Deep Sequencing of Porphyromonas gingivalis and comparative transcriptome analysis of a LuxS mutant

    Takanoi eHirano

    2012-06-01

    Full Text Available Porphyromonas gingivalis is a major etiological agent and chronic and aggressive forms of periodontal disease. The organism is an assacharolytic anaerobe and is a constituent of mixed species biofilms in a variety of microenvironments in the oral cavity. P. gingivalis expresses a range of virulence factors over which it exerts tight control. High-throughput sequencing technologies provide the opportunity to relate functional genomics to basic biology. In this study we report qualitative and quantitative RNA-Seq analysis of the transcriptome of P. gingivalis. We have also applied RNA-Seq to the transcriptome of a ΔluxS mutant of P. gingivalis deficient in AI-2-mediated bacterial communication. The transcriptome analysis confirmed the expression of all predicted ORFs for strain ATCC 33277, including 854 hypothetical proteins, and allowed the identification of hitherto unknown transcriptional units. Twelve noncoding RNAs were identified, including 11 small RNAs and one cobalamine riboswitch. Fifty seven genes were differentially regulated in the LuxS mutant. Addition of exogenous synthetic 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor to the ΔluxS mutant culture complemented expression of a subset of genes, indicating that LuxS is involved in both AI-2 signaling and non-signaling dependent systems in P. gingivalis. This work provides an important dataset for future study of P. gingivalis pathophysiology and further defines the LuxS regulon in this oral pathogen.

  3. Exploratory and discriminative studies of commercial processed Brazilian coffees with different degrees of roasting and decaffeinated

    Juliano Souza Ribeiro

    2013-09-01

    Full Text Available The fingerprints of the volatile compounds of 21 commercial Brazilian coffee samples submitted to different industrial processing i.e. decaffeinated or different roasting degrees (traditional and dark were studied. The volatiles were collected by headspace solid phase microextraction (HS-SPME and analyzed by GC-FID and GC-MS. The chromatographic data matrices (fingerprints obtained were explored by the principal component analysis (PCA and partial least squares - discriminative analysis (PLS-DA. Initially the chromatographic profiles were aligned by the algorithm correlation optimized warping (COW. The PCA showed the discrimination of the decaffeinated coffees from the others with both the SPME fibres used. This separation probably occurred due to the loss of some volatile precursors during the decaffeination process, such as sucrose. For both the fibres tested, PDMS/DVB and CX / PDMS SPME, the PLS-DA models correctly classified 100% of the samples according to their roasting degree: (medium and dark, the main differences being the concentrations of some of the volatile compounds such as 2-methyl furan, 2-methylbutanal, 2,3-pentanedione, pyrazine, 2-carboxyaldehyde pyrrole, furfural and 2-furanmethanol.

  4. Characterisation of lactic acid bacteria isolated from fermented milk "laban".

    Chammas, Gisele I; Saliba, Rachad; Corrieu, Georges; Béal, Catherine

    2006-07-01

    The technological properties of 96 lactic acid bacteria isolated from Lebanese traditional fermented milk "laban" were characterised. They were classified by phenotypic and biochemical analyses as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, thus indicating that laban is a fermented milk similar to yogurt. Most strains of L. bulgaricus (87.5%) exhibited a high acidification activity, whereas strains of streptococci showed low acidification ability. 33.3% of streptococci strains and 25% of lactobacilli strains displayed similar acidification performances as European strains. Results obtained for syneresis, texture and rheological parameters led us to consider that isolated strains were not low polymer-producing strains. Some of them displayed interesting characteristics such as low syneresis and high values for rheological parameters. The major flavour compounds found in pure cultures were acetaldehyde, acetone, 2-butanone, dimethyl disulfide, acetoin, 2,3-butanedione, 2,3-pentanedione, and acetic, hexanoic and butanoic acids. Acetaldehyde (7.4%) and organic acids (48.3%) were mainly produced by L. bulgaricus strains, whereas streptococci cultures contained high relative levels of 2,3-butanedione and acetoin, which represented around 82% of the total flavour compounds. Finally, strains isolated from laban samples exhibited different technological properties than those used in yogurt production, thus conferring specific characteristics to this product.

  5. Characterization of the key odorants in raw Italian hazelnuts ( Corylus avellana L. var. Tonda Romana) and roasted hazelnut paste by means of molecular sensory science.

    Burdack-Freitag, Andrea; Schieberle, Peter

    2012-05-23

    The concentrations of 19 odorants, recently characterized by GC-olfactometry and aroma extract dilution analysis as the most odor-active compounds in raw hazelnuts, were quantitated by stable isotope dilution assays (SIDA). Calculation of odor activity values (OAV) on the basis of odor thresholds in oil revealed high OAVs, in particular for linalool, 5-methyl-4-heptanone, 2-methoxy-3,5-dimethylpyrazine, and 4-methylphenol. A model mixture in sunflower oil containing the 13 odorants showing OAVs above 1 in their natural concentrations resulted in a good similarity compared to the overall nut-like, fruity aroma of the raw hazelnuts. Quantitation of the 25 most odor-active compounds in roasted hazelnut paste by SIDA showed clear changes in the concentrations of most odorants, and formation of new odor-active compounds induced by the roasting process was observed. The highest OAVs were calculated for 3-methylbutanal (malty), 2,3-pentanedione (buttery), 2-acetyl-1-pyrroline (popcorn), and (Z)-2-nonenal (fatty), followed by dimethyl trisulfide, 2-furfurylthiol, 2,3-butanedione, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone. The aroma of a model mixture containing the 19 odorants with OAVs above 1 in their actual concentrations in the roasted nut material was judged to elicit a very good similarity to the popcorn-like, coffee-like, and sweet-smoky aroma of the roasted hazelnut paste. New SIDAs were developed for the quantitation of 5-methyl-4-heptanone, 5-methyl-(E)-2-hepten-4-one, 2-thenylthiol, and 3,5,5-trimethyl-2(5H)-furanone.

  6. Processing the Interspecies Quorum-sensing Signal Autoinducer-2 (AI-2)

    J Marques; P Lamosa; C Russell; R Ventura; C Maycock; M Semmelhack; S Miller; K Xavier

    2011-12-31

    The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.

  7. Multi-volatile method for aroma analysis using sequential dynamic headspace sampling with an application to brewed coffee.

    Ochiai, Nobuo; Tsunokawa, Jun; Sasamoto, Kikuo; Hoffmann, Andreas

    2014-12-05

    A novel multi-volatile method (MVM) using sequential dynamic headspace (DHS) sampling for analysis of aroma compounds in aqueous sample was developed. The MVM consists of three different DHS method parameters sets including choice of the replaceable adsorbent trap. The first DHS sampling at 25 °C using a carbon-based adsorbent trap targets very volatile solutes with high vapor pressure (>20 kPa). The second DHS sampling at 25 °C using the same type of carbon-based adsorbent trap targets volatile solutes with moderate vapor pressure (1-20 kPa). The third DHS sampling using a Tenax TA trap at 80 °C targets solutes with low vapor pressure (aroma compounds for each DHS sampling and the combined MVM procedure were evaluated as a function of vapor pressure in the range of 0.000088-120 kPa. The MVM provided very good recoveries in the range of 91-111%. The method showed good linearity (r2>0.9910) and high sensitivity (limit of detection: 1.0-7.5 ng mL(-1)) even with MS scan mode. The feasibility and benefit of the method was demonstrated with analysis of a wide variety of aroma compounds in brewed coffee. Ten potent aroma compounds from top-note to base-note (acetaldehyde, 2,3-butanedione, 4-ethyl guaiacol, furaneol, guaiacol, 3-methyl butanal, 2,3-pentanedione, 2,3,5-trimethyl pyrazine, vanillin, and 4-vinyl guaiacol) could be identified together with an additional 72 aroma compounds. Thirty compounds including 9 potent aroma compounds were quantified in the range of 74-4300 ng mL(-1) (RSD<10%, n=5).

  8. Quorum sensing signal production and microbial interactions in a polymicrobial disease of corals and the coral surface mucopolysaccharide layer.

    Beth L Zimmer

    Full Text Available Black band disease (BBD of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs and for autoinducer-2 (AI-2 activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153 of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S-4,5-dihydroxy-2,3-pentanedione (DPD, the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.

  9. Elucidation of the conformational free energy landscape in H.pylori LuxS and its implications to catalysis

    Bhattacharyya Moitrayee

    2010-08-01

    Full Text Available Abstract Background One of the major challenges in understanding enzyme catalysis is to identify the different conformations and their populations at detailed molecular level in response to ligand binding/environment. A detail description of the ligand induced conformational changes provides meaningful insights into the mechanism of action of enzymes and thus its function. Results In this study, we have explored the ligand induced conformational changes in H.pylori LuxS and the associated mechanistic features. LuxS, a dimeric protein, produces the precursor (4,5-dihydroxy-2,3-pentanedione for autoinducer-2 production which is a signalling molecule for bacterial quorum sensing. We have performed molecular dynamics simulations on H.pylori LuxS in its various ligand bound forms and analyzed the simulation trajectories using various techniques including the structure network analysis, free energy evaluation and water dynamics at the active site. The results bring out the mechanistic details such as co-operativity and asymmetry between the two subunits, subtle changes in the conformation as a response to the binding of active and inactive forms of ligands and the population distribution of different conformations in equilibrium. These investigations have enabled us to probe the free energy landscape and identify the corresponding conformations in terms of network parameters. In addition, we have also elucidated the variations in the dynamics of water co-ordination to the Zn2+ ion in LuxS and its relation to the rigidity at the active sites. Conclusions In this article, we provide details of a novel method for the identification of conformational changes in the different ligand bound states of the protein, evaluation of ligand-induced free energy changes and the biological relevance of our results in the context of LuxS structure-function. The methodology outlined here is highly generalized to illuminate the linkage between structure and function in

  10. Identification of aroma active compounds of cereal coffee brew and its roasted ingredients.

    Majcher, Małgorzata A; Klensporf-Pawlik, Dorota; Dziadas, Mariusz; Jeleń, Henryk H

    2013-03-20

    Cereal coffee is a coffee substitute made mainly from roasted cereals such as barley and rye (60-70%), chicory (15-20%), and sugar beets (6-10%). It is perceived by consumers as a healthy, caffeine free, non-irritating beverage suitable for those who cannot drink regular coffee made from coffee beans. In presented studies, typical Polish cereal coffee brew has been subjected to the key odorants analysis with the application of gas chromatography-olfactometry (GC-O) and aroma extract dilution analysis (AEDA). In the analyzed cereal coffee extract, 30 aroma-active volatiles have been identified with FD factors ranging from 16 to 4096. This approach was also used for characterization of key odorants in ingredients used for the cereal coffee production. Comparing the main odors detected in GC-O analysis of roasted cereals brew to the odor notes of cereal coffee brew, it was evident that the aroma of cereal coffee brew is mainly influenced by roasted barley. Flavor compound identification and quantitation has been performed with application of comprehensive multidimentional gas chromatography and time-of-flight mass spectrometry (GCxGC-ToFMS). The results of the quantitative measurements followed by calculation of the odor activity values (OAV) revealed 17 aroma active compounds of the cereal coffee brew with OAV ranging from 12.5 and 2000. The most potent odorant was 2-furfurylthiol followed by the 3-mercapto-3-methylbutyl formate, 3-isobutyl-2-methoxypyrazine and 2-ethyl-3,5-dimethylpyrazine, 2-thenylthiol, 2,3-butanedione, 2-methoxy phenol and 2-methoxy-4-vinyl phenol, 3(sec-butyl)-2-methoxypyrazine, 2-acetyl-1-pyrroline, 3-(methylthio)-propanal, 2,3-pentanedione, 4-hydroxy-2,5-dimethyl-3-(2H)-furanone, (E,E)-2,4-decadienal, (Z)-4-heptenal, phenylacetaldehyde, and 1-octen-3-one.

  11. Quorum sensing signals affect spoilage of refrigerated large yellow croaker (Pseudosciaena crocea) by Shewanella baltica.

    Zhu, Junli; Zhao, Aifei; Feng, Lifang; Gao, Haichun

    2016-01-18

    In this work we investigated the specific spoilage organism (SSO) of large yellow croaker (Pseudosciaena crocea) stored at 4°C and role of quorum sensing (QS) system of SSO isolated from the spoiled fish. According to microbial count and 16S rRNA gene of the isolated pure strains, Shewanella, mainly Shewanella baltica and Shewanella putrefaciens, was predominant genera at the end of shelf-life of P. crocea. Among Shewanella isolates, S.baltica02 was demonstrated as SSO in spoilage potential characteristics by inoculation into sterile fish juice using sensory and chemical analyses. Autoinducer 2 and two cyclic dipeptides (DKPs) including cyclo-(l-Pro-l-Leu) and cyclo-(l-Pro-l-Phe), no any AHLs, were detected in cell-free S. baltica culture. Interestingly, S.baltica02 had the highest QS activity among three spoilers of S. baltica. The production of biofilm, trimethylamines (TMA) and putrescine in these spoilers significantly increased in the presence of cyclo-(l-Pro-l-Leu), rather than cyclo-(l-Pro-l-Phe) and 4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor, DPD). In accordance with the effect of signal molecules on the spoilage phenotype, exposure to exogenous cyclo-(l-Pro-l-Leu) was also showed to up-regulate the transcription levels of luxR, torA and ODC, and no effect of luxS indicated that S. baltica could sense cyclo-(l-Pro-l-Leu). In the fish homogenate, exogenous cyclo-(l-Pro-l-Leu) shortened lag phase durations and enhanced growth rates of the dominant bacteria, H2S producing bacteria, under refrigerated storage, while exogenous DPD retarded growth of competing bacteria, such as Enterobacteriaceae. Meanwhile, cyclo-(l-Pro-l-Leu) also promoted the accumulation of metabolites on the spoilage process of homogenate. S.baltica02 luxS mutant preliminarily proved that AI-2 might not play a signaling role in the spoilage. The present study suggested that the spoilage potential of S. baltica in P. crocea might be regulated by DKP-based quorum sensing.

  12. Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase.

    Calam, Eduard; González-Roca, Eva; Fernández, M Rosario; Dequin, Sylvie; Parés, Xavier; Virgili, Albert; Biosca, Josep A

    2016-03-01

    Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms-purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)-to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.

  13. Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase

    Calam, Eduard; González-Roca, Eva; Fernández, M. Rosario; Dequin, Sylvie; Parés, Xavier; Virgili, Albert

    2016-01-01

    Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction. PMID:26729717

  14. In Helicobacter pylori auto-inducer-2, but not LuxS/MccAB catalysed reverse transsulphuration, regulates motility through modulation of flagellar gene transcription

    Doherty Neil

    2010-08-01

    Full Text Available Abstract Background LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2; hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB, synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only. Results We report that disruption of luxS renders H. pylori non-motile in soft agar and by microscopy, whereas disruption of mccAHp or mccBHp (other genes in the cysteine provision pathway does not, implying that the lost phenotype is not due to disrupted cysteine provision. The motility defect of the ΔluxSHp mutant was complemented genetically by luxSHp and also by addition of in vitro synthesised AI-2 or 4, 5-dihydroxy-2, 3-pentanedione (DPD, the precursor of AI-2. In contrast, exogenously added cysteine could not restore motility to the ΔluxSHp mutant, confirming that AI-2 synthesis, but not the metabolic effect of LuxS was important. Microscopy showed reduced number and length of flagella in the ΔluxSHp mutant. Immunoblotting identified decreased levels of FlaA and FlgE but not FlaB in the ΔluxSHp mutant, and RT-PCR showed that the expression of flaA, flgE, motA, motB, flhA and fliI but not flaB was reduced. Addition of DPD but not cysteine to the ΔluxSHp mutant restored flagellar gene transcription, and the number and length of flagella. Conclusions Our data show that as well as being a metabolic enzyme, H. pylori LuxS has an alternative role in regulation of motility by modulating flagellar transcripts and flagellar biosynthesis through production of the signalling molecule AI-2.