The formation mechanism by yeast of 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone in Miso

The mechanism of the formation of 4-hydroxy-2(or 5)-ethyl-5-(or 2)-methyl-3(2H)-furanone (HEMF) with yeast under caltivation in a medium containing amino-carbonyl reactants of ribose and glycine was investigated using stable isotopes of the corresponding compounds. It was confirmed that the skeleton of the five-membered ring and the methyl group of the side chain of HEMF was formed from ribose, and that the ethyl group was derived from the glucose metabolite by yeast. The formation of HEMF was confirmed when acetaldehyde as the glucose metabolite and a cell-free extract from yeast were added to the medium containing amino-carbonyl reactants. These results suggest that the role of yeast in HEMF formation is not only to provide the glucose metabolite, but also in combining the amino-carbonyl reactants with the glucose metabolite.     

Effect of media constituents on the formation by halophilic yeast of the 2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone aroma component specific to miso.

The formation of HEMF [2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone] by yeast was examined in an attempt to investigate its mechanism and involved factors. HEMF formation was promoted by yeast cultivation in a heat-sterilized medium which included glucose, ribose, and a nitrogenous compound such as an extract of shoyu koji, poly-peptone, casamino acid, or an amino acid (glutamic acid, threonine, serine, or alanine).     

Evaluation of multiwavelength culture fluorescence for monitoring the aroma compound 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) production

Fluorescence spectra of a 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) fermentation culture broth were combined with measurable process variables for off-line and on-line process monitoring. Culture broth fluorescence in UV and visible ranges was acquired by a fiber optic LCD array spectrometer. Process dynamics was followed on-line using a fiber optic probe attached to an external recirculation loop of the bioreactor. Partial least squares and stepwise regression methods were used to correlate measurable process parameters with the components of the fluorescence spectra. Both methods provided adequate approximation of yeast density, HEMF, glucose, and ethanol concentrations from fluorescence spectra. HEMF production was observed during the oxido-reductive growth phase when there was a lack of measurable oxygen in the culture broth and an excess of glucose. The addition of glucose resulted in the rapid production of HEMF and other metabolite intermediates such as ethanol, acetate, and glycerol.     

Screening of High-Level 4-Hydroxy-2 (or 5)-Ethyl-5 (or 2)-Methyl-3(2H)-Furanone-Producing Strains from a Collection of Gene Deletion Mutants of Saccharomyces cerevisiae

4-Hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone (HEMF) is an important flavor compound that contributes to the sensory properties of many natural products, particularly soy sauce and soybean paste. The compound exhibits a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. HEMF is produced by yeast species in soy sauce manufacturing; however, the enzymes involved in HEMF production remain unknown, hindering efforts to breed yeasts with high-level HEMF production. In this study, we identified high-level HEMF-producing mutants among a Saccharomyces cerevisiae gene deletion mutant collection. Fourteen deletion mutants were screened as high-level HEMF-producing mutants, and the ADH1 gene deletion mutant (adh1Δ) exhibited the maximum HEMF production capacity. Further investigations of the adh1Δ mutant implied that acetaldehyde accumulation contributes to HEMF production, agreeing with previous findings. Therefore, acetaldehyde might be a precursor for HEMF. The ADH1 gene deletion mutant of Zygosaccharomyces rouxii, which is the dominant strain of yeast found during soy sauce fermentation, also produces HEMF effectively, suggesting that acetaldehyde accumulation might be a benchmark for breeding industrial yeasts with excellent HEMF production abilities.     

Formation by Yeast of the HEMF (4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone) Aroma Component in Miso with Aging

Two kinds of miso, one with added precultured yeast and the other without, were compared with respect to the changes in the concentration of HEMF formed and the number of yeast cells in the process of aging. In miso without added yeast, the HEMF concentration increased with the increasing number of existing yeast cells. In miso without yeast aged for 21 days after the miso mash, 0.06 ppm HEMF was detected when the cell number was 2.2 × 10³ cell/g. In yeast-added miso aged for 7 days after the miso mash, no HEMF was detected, although the number of yeast cells was 1.6 × 10⁶ cell/g. In yeast-added miso aged for 14 days after the miso mash, HEMF was first detected. The pH levels of miso without yeast and with added yeast when HEMF was first detected were 5.59 and 5.57, respectively. It is suggested that the formation of HEMF in miso containing a high concentration of reducing sugar and salt was related to the growth of yeast and started when the pH level fell to less than 5.6.     

Formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone by Zygosaccharomyces rouxii: identification of an intermediate

The formation of the important flavor compound 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDMF; Furaneol) from D-fructose-1,6-bisphosphate by the yeast Zygosaccharomyces rouxii was studied with regard to the identification of intermediates present in the culture medium. Addition of o-phenylenediamine, a trapping reagent for alpha-dicarbonyls, to the culture medium and subsequent analysis by high-pressure liquid chromatography with diode array detection revealed the formation of three quinoxaline derivatives derived from D-fructose-1,6-bisphosphate under the applied growth conditions (30 degrees C; pH 4 to 5). Isolation and characterization of these compounds by tandem mass spectrometry and nuclear magnetic resonance spectroscopy led to the identification of phosphoric acid mono-(2,3,4-trihydroxy-4-quinoxaline-2-yl-butyl) ester (Q1), phosphoric acid mono-[2,3-dihydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q2), and phosphoric acid mono-[2-hydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q3). Q1 and Q2 were formed independently of Z. rouxii cells, whereas Q3 was detected only in incubation systems containing the yeast. Identification of Q2 demonstrated for the first time the chemical formation of 1-deoxy-2,3-hexodiulose-6-phosphate in the culture medium, a generally expected but never identified intermediate in the formation pathway of HDMF. Since HDMF was detected only in the presence of Z. rouxii cells, additional enzymatic steps were presumed. Incubation of periplasmic and cytosolic protein extracts obtained from yeast cells with D-fructose-1,6-bisphosphate led to the formation of HDMF, implying the presence of the required enzymes in both extracts.     

Effect of food reductones on the generation of the pyrazine cation radical and on the formation of the mutagens in the reaction of glucose, glycine and creatinine

One of the possible pathways of the formation of mutagens in heated foods is through the pyrazine cation radical generated in the early stage of the Maillard reaction. The aim of the present study was to elucidate how food reductones contribute to the pyrazine cation radical generation in the reaction of glucose (Glc) and glycine (Gly), and to the formation of the mutagens in the reaction of Glc, Gly and creatinine. Electron spin resonance (ESR) studies showed that fragrant reductones, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), generated in the Maillard reactions, enhanced the generation of the pyrazine cation radical in the reaction of Glc and Gly, and the reaction of DMHF or HEMF with Gly generated a larger amount of the pyrazine cation radical than the reaction of Glc and Gly, indicating that the furanones were intermediates of the pyrazine cation radical. By contrast, food antioxidants, ascorbic acid and erythorbic acid, effectively scavenged the pyrazine cation radical generated in the reaction of Glc and Gly. DMHF and HEMF were not effective to modulate the mutagen formation in the reaction of Glc, Gly and creatinine, and the mutagenicity produced in the reaction of DMHF or HEMF, Gly and creatinine was lower than that produced in the reaction of Glc, Gly and creatinine. On the other hand, ascorbic acid and erythorbic acid were effective to decrease the mutagen formation in the reaction of Glc, Gly and creatinine.     

Tautomerism of 4-hydroxy-2,5-dimethyl-3(2H)-furanone: evidence for its enantioselective biosynthesis

Chiral natural flavor compounds exhibit characteristic enantiomeric excesses due to stereoselective, enzymatically catalyzed reactions during biogenesis. Although the enzymatic formation of the strawberry key flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol(R)) is anticipated, the naturally occurring compound is racemic. As racemization due to keto-enol-tautomerism of HDMF could account for this observation, HDMF was investigated by (1)H-NMR spectroscopy tracing the exchange of the proton bound to the furanone-ring at C2 with deuteron from the medium (D(2)O). In addition, the racemization rate of HDMF was directly determined by cyclodextrin-modified capillary electrophoresis of enantiomerically enriched HDMF stored at different pH values. Tautomerism and the racemization rate of HDMF was lowest at pH values between 4 and 5. However, tautomerism and thus racemization was catalyzed under stronger acidic conditions (pH 2) and especially at pH values greater than 7, the value published for plant cell cytosol. Approximately 50% of the protons at C2 were exchanged with deuteron within 1 h at pH 7.2. Therefore, in order to demonstrate the enzymatic formation of HDMF, incubation experiments with Zygosaccharomyces rouxii as well as strawberry protein extract were carried out under slightly acidic conditions (pH 5), the most suitable pH value for studies on the enantiomeric ratio of HDMF. In both experiments the formation of enantiomerically enriched HDMF could be demonstrated for the first time, whereas incubation experiments under neutral conditions resulted in the detection of racemic HDMF.     

Biochemical roles of the oligosaccharide chains in thyrostimulin, a heterodimeric hormone of glycoprotein hormone subunits alpha 2 (GPA2) and beta 5 (GPB5)

Thyrostimulin is a heterodimeric hormone composed of GPA2 and GPB5, and shares the thyroid-stimulating hormone receptor (TSHR). Thyrostimulin has three N-linked oligosaccharide chains, two in GPA2 and one in GPB5. The roles of these N-linked oligosaccharides in secretion, heterodimer formation and signal transduction were analyzed. Recombinant GPA2s lacking either of the two oligosaccharides were obtained from conditioned medium, whereas dual site-disrupted GPA2 and the GPB5 mutant were not expressed in either the conditioned medium or cell lysate. The binding between GPA2 and GPB5 was weaker than that between TSH subunits GPA1 and TSH beta. Neither of the oligosaccharides in GPA2 had significant effects on heterodimerization. Disruption of either of the oligosaccharides in GPA2 significantly decreased receptor activation, suggesting their critical role in receptor activation.     

Biosynthesis of flavour-active furanones by Saccharomyces cerevisiae during fermentation depends on the malt type used in medium preparation

Extracts of a coloured malt contained 4-hydroxy-5-monomethyl-3(2H)-furanone (HMMF), 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) but not 4-hydroxy-5(or 2)-ethyl-2(or 5)-methyl-3(2H)-furanone (HEMF). Extracts of a pale malt did not contain any of the furanones. HMMF and HDMF were produced by Saccharomyces cerevisiae during fermen-tation of both types of malt extract. About 0.09 mg HEMF l ⁻¹ was synthesised during fermentation of the coloured malt extract but none was produced with the pale malt extract. Final concentrations of HDMF (2.0 mgl ⁻¹) and HEMF (0.09 mgl ⁻¹) were in excess of their aroma threshold values in water (0.16 and 0.02 mgl ⁻¹ respectively) after fermen-tation of the coloured malt extract. This revised version was published online in November 2006 with corrections to the Cover Date.     

Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.     

Efficient triple helix formation by oligodeoxyribonucleotides containing alpha- or beta-2-amino-5-(2-deoxy-D-ribofuranosyl) pyridine residues.

Triple helices containing C+xGxC triplets are destabilised at physiological pH due to the requirement for base protonation of 2'-deoxycytidine (dC), which has a pKa of 4.3. The C nucleoside 2-amino-5-(2'-deoxy-beta-D-ribofuranosyl)pyridine (beta-AP) is structurally analogous to dC but is considerably more basic, with a pKa of 5.93. We have synthesised 5'-psoralen linked oligodeoxyribonucleotides (ODNs) containing thymidine (dT) and either beta-AP or its alpha-anomer (alpha-AP) and have assessed their ability to form triplexes with a double-stranded target derived from standard deoxynucleotides (i.e. beta-anomers). Third strand ODNs derived from dT and beta-AP were found to have considerably higher binding affinities for the target than the corresponding ODNs derived from dT and either dC or 5-methyl-2'-deoxycytidine (5-Me-dC). ODNs containing dT and alpha-AP also showed enhanced triplex formation with the duplex target and, in addition are more stable in serum-containing medium than standard oligopyrimidine-derived ODNs or ODNs derived from dT and beta-AP. Molecular modelling studies showed that an alpha-anomeric AP nucleotide can be accommodated within an otherwise beta-anomeric triplex with only minor perturbation of the triplex structure. Molecular dynamics (MD) simulations on triplexes containing either the alpha- or beta-anomer of (N1-protonated) AP showed that in both cases the base retained two standard hydrogen bonds to its associated guanine when the 'A-type' model of the triplex was used as the start-point for the simulation, but that bifurcated hydrogen bonds resulted when the alternative 'B-type' triplex model was used. The lack of a differential stability between alpha-AP- and beta-AP-containing triplexes at pH >7, predicted from the behaviour of the B-type models, suggests that the A-type models are more appropriate.     

Transformation of 3beta-(2-hydroxyethoxy)-5alpha-cholest-8(14)-en-15-one in a polar metabolite in HepG2 hepatoma cells

Incubation of 3 beta-(2-hydroxy-2[3H]-ethoxy)-5 alpha-cholest-8(14)-en-15-one with Hep G2 cells led to the accumulation of a radioactive polar product in the culture medium, which was identified as 3 beta-(2-hydroxyethoxy)-15-keto-5 alpha-cholest-8(14)-ene-24-oic acid. Its structure was confirmed by a chemical counter synthesis. The labeled ketosterol was rapidly (tau 1/2 = 6 min) and reversibly bound by Hep G2 cells. The intracellular concentration of 15-ketosterol decreased during incubation mainly due to the formation of a polar metabolite, secreted to the medium. The level of cholesterol biosynthesis was 22 +/- 5% of the control value in Hep G2 cells at a 15-ketocholesterol concentration in the medium of 30 microM. However, further incubation for 3 h in the medium without the ketosterol led to restoration of the level of biosynthesis to 85 +/- 11% of the control value. These results suggest that inhibition of the cholesterol biosynthesis by 15-ketocholesterol in Hep G2 cells depends on the intracellular concentration of the inhibitor, which, in turn, is determined by the rate of its conversion into the polar metabolite. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

A convenient synthesis of adenylyl-(2'----5')-adenylyl-(2'----5')-adenosine (2-5A core)

A simple synthesis of adenylyl-(2'----5')-adenylyl (2'----5')-adenosine (2-5A core) has been achieved on the basis of selective 3'-O-silylation of 5'-O-p-monomethoxytrityladenosine and chemo-selective formation of the 2'-5' internucleotide linkage using N-unprotected nucleosides.     

Microbial water relations. Effects of solute concentration on the respiratory activity of sugar-tolerant and non-tolerant yeasts.

The respiratory activity of the sugar-tolerant (osmophilic) yeast, Saccharomyces rouxii, and the non-tolerant species, Sacchromyces cerevisiae, were compared after growth in a complex basal medium, the medium supplemented with polyethylene glycol (mol. wt 200) to give a water activity of 0-95, and the medium supplemented with glucose (24 and 36%, w/v). The properties compared were Qo2 (glucose), NADH oxidase activity of isolated mitochondrial fractions, and cytochrome content. When grown in the basal medium S. cerevisiae was somewhat more active than S. rouxii by all criteria. Growth in the media supplemented were high glucose concentrations produced catabolite repression of respiration in S. cerevisiae but not in S. rouxii. The implications of this difference for polyol biosynthesis and the water relations of the sugar-tolerant species are discussed.     

Effect of nystatin on the release of glycerol from salt-stressed cells of the salt-tolerant yeast Zygosaccharomyces rouxii

The polyene antibiotic nystatin, which affects fungal membrane permeability, inhibited the growth of Zygosaccharomyces rouxii grown in medium containing 15% (w/v) NaCl, whereas yeast grown in medium without NaCl were only slightly inhibited. Nystatin caused salt-stressed cells to release large amounts of glycerol and inhibited their growth, but amino acids and materials with an absorbance at 260 nm were not released from the cells. The leakage was increased by the addition of glucose, and more than 90% of the intracellular glycerol was released into the medium during a 2-h incubation with 0.11 microM nystatin and 2% (w/v) glucose. Glycerol was indispensable for the growth of Z. rouxii grown in culture medium containing 15% NaCl.     

Synthesis and antimetabolite properties of 5-substituted 2'-deoxyuridines. Anomeric 5-(2-aminohexafluoroprop-2-yl)- and 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)-2'-deoxyuridine

Alkylation of 2,4-bis-O-(trimethylsilyl)uracil with hexafluoroacetone trifluoroacetylimine gave 5-(2-trifluoroacelylaminohexafluoroprop-2-yl)uracil, which was transformed by alkaline hydrolysis to 5-(2-aminohexafluoroprop-2-yl)uracil. The latter was glycosytated with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-ribofyranosyl chloride by means of various modifications of the silyl method leading to the predominant formation of beta-deoxynucleoside; after deacylation 1-(2-deoxy-beta-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ura cil was obtained. Interaction of silylated 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)uracil with acylgalogenose gave anomeric O-substitutet deoxynucleosides, which were deblocked to give 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)-2'-deoxyuridine and corresponding alpha-anomer. Alkaline hydrolysis of N-trifluoroacetyl group in both individual anomers produced 1-(2-deoxy-alpha-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ur acil and the abovementioned beta-anomer. Of all compounds synthesised only 1-(2-deoxy-beta-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ura cil has a moderate inhibitory effect on replication of vaccinia virus in vitro.     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.