End Products of Glucose Fermentation by Brochothrix thermosphacta

Anaerobically, Brochothrix thermosphacta fermented glucose primarily to l-lactate, acetate, formate, and ethanol. The ratio of these end products varied with growth conditions. Both the presence of acetate and formate and a pH below about 6 increased l-lactate production from glucose. Small amounts of butane-2,3-diol were also produced when the pH of the culture was low (相似文献   

Regulation of heart muscle pyruvate dehydrogenase kinase

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).     

The metabolism of glyoxylate by cell-free extracts of Pseudomonas sp

1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-¹⁴C]glyoxylate. 3. The ¹⁴C was incorporated from [1-¹⁴C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The ¹⁴C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase.     

Synthesis of A New Intercalating Nucleic Acid 6H-INDOLO[2,3-b] Quinoxaline Oligonucleotides to Improve Thermal Stability Of Hoogsteen-Type Triplexes

A new intercalating nucleic acid monomer X was obtained in high yield starting from alkylation of 4-iodophenol with (S)-(+)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol under Mitsunobu conditions followed by hydrolysis with 80% aqueous acetic acid to give a diol which was coupled under Sonogashira conditions with trimethylsilylacetylene (TMSA) to achieve the TMS protected (S)-4-(4-((trimethylsilyl)ethynyl)phenoxy)butane-1,2-diol. Tetrabutylammonium flouride was used to remove the silyl protecting group to obtain (S)-4-(4-ethynylphenoxy)butane-1,2-diol which was coupled under Sonogashira conditions with 2-(9-bromo-6H-indolo[2,3-b]quinoxalin-6-yl)-N,N-dimethylethanamine to achieve (S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[2,3-b]quinoxalin-9-yl)ethynyl)phenoxy)butane-1,2-diol. This compound was tritylated with 4,4′-dimethoxytrityl chloride followed by treatment with 2-cyanoethyltetraisopropylphosphordiamidite in the presence of N,N′-diisopropyl ammonium tetrazolide to afford the corresponding phosphoramidite. This phosphoramidite was used to insert the monomer X into an oligonucleotide which was used for thermal denaturation studies of a corresponding parallel triplex.     

The effect of pH and temperature on haemolysin production by Listeria species

Acetoin reductase (EC 1.1.1.4) from Kluyveromyces marxianus var. marxianus NRRL Y-1196 was found to possess the highest specific activity (3.64 units/mg protein) of the four cultures studied. The enzyme was NADH-dependent and catalysed the conversion of acetoin to 2,3-butanediol. It was stable at 40°C for 30 min, but lost 50% cf its activity after 15 min at 50°C. The optimum pH for the enzymatic reduction of acetoin was 7.0. The K _m values of the crude enzyme for acetoin and NADH were determined to be 0.57 mmol/l and 0.045 mmol/l, respectively.     

Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.Acetoin and 2,3-butanediol are minor products generated by Saccharomyces cerevisiae during alcohol fermentation. Their sensory impacts on wine are poorly documented. Acetoin may affect the wine bouquet, although its perception threshold in wine is relatively high, around 150 mg/liter (21, 31). On the other hand, 2,3-butanediol is odorless (33) and cannot be expected to appreciably affect the sensory quality of wine. However, the compound may contribute to the wine body (28).Acetaldehyde, pyruvate, and α-acetolactate are the main precursors of acetoin in S. cerevisiae. Acetoin can be formed from acetaldehyde and/or pyruvate through an anomalous reaction of pyruvate decarboxylase. Thus, although its main activity is to irreversibly decarboxylate pyruvate to acetaldehyde, it can also catalyze carbon-carbon bond formation, yielding acetoin from pyruvate and/or acetaldehyde (2, 4). In addition, α-acetolactate would produce acetoin through its nonenzymatic decarboxylation to diacetyl and subsequent reduction to acetoin through the action of several NADH- and NADPH-dependent oxidoreductases (12). However, the situation is more complex in wine fermentation, where other yeasts and bacteria display supplementary enzymatic activities capable of producing both acetoin and 2,3-butanediol (1, 27).We have previously characterized a butanediol dehydrogenase (Bdh1p) as a medium-chain dehydrogenase/reductase (MDR) that can reversibly transform R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively, in a NAD(H)-dependent reaction (10). BDH2 is a gene adjacent to BDH1 whose uncharacterized protein product (Bdh2p) shares 51% sequence identity with Bdh1p. To evaluate the in vivo roles of Bdh1p and Bdh2p, we compared the levels of several extracellular metabolites in cultures of wild-type and deficient strains. The results show that, although Bdh1p is the main enzyme in 2,3-butanediol production [essentially the (2R,3R)-2,3-butanediol stereoisomer], some meso-2,3-butanediol is still produced by the bdh1Δ strains. We have characterized Ara1p as an oxidoreductase that can reduce racemic acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol in the presence of NADPH.Furthermore, we have overexpressed Bdh2p with a histidine tag at its carboxyl terminus and have shown it to be inactive toward acetoin and 2,3-butanediol. A microarray study indicated that BDH1 and BDH2 are reciprocally regulated under the conditions studied.     

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5''-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.     

增强胞内NDAH水平和乙偶姻还原酶活力提高2,3-丁二醇产量

枯草芽孢杆菌Bacillus subtilis 168是一株安全生产菌株,首次通过弱化B.subtilis 168磷酸戊糖途径(PPP)中的关键酶葡萄糖-6-磷酸脱氢酶(G6PDH)基因zwf,研究了其对胞内NADH水平的影响,进而研究其对2,3-丁二醇(2,3-BD)及副产物合成的影响。弱化菌株B. subtilis168△zwf进行摇瓶发酵实验,与出发菌株相比,胞内辅酶NADH水平得到了增强, 2,3-BD产量提高了15.0%,主要副产物AC积累量下降了10.6%,但乙酸、乳酸等有机酸的积累量提高。为了进一步提高2,3-BD生产效率,在B. subtilis168中克隆表达了不同来源的ACR基因,研究发现克雷伯氏菌来源的ACR酶活力最高,将此来源的ACR的基因kphs克隆到B.subtilis168△zwf中加强表达,对重组菌株B.subtilis168△zwf/pMA5-kphs进行摇瓶发酵实验,与出发菌相比,2,3-BD产量提高了37.3 %,主要副产物AC积累量下降了28.1%,同时,乙酸等分支路径的其他副产物也有不同程度的降低。     

Effect of thiamine on the activity of the pyruvate dehydrogenase complex in rat liver following stimulation of lipogenesis

It is shown that thiamine administration to rats (250 micrograms per 100 g of mass) who were given high-carbohydrate diet (lipogenesis intensification) after fasting inhibits an increase in the pyruvate dehydrogenase activity in the liver homogenate and mitochondria usual under these conditions. This is observed when determining total activity of the pyruvate dehydrogenase complex and activity of its first component--pyruvate dehydrogenase estimated from the ferricyanide reduction and [1-14C] CO2 formation from [1-14C] pyruvate. Fasting animals and animals whom thiamine was administered against a background of lipogenesis intensification revealed a higher ability of the liver tissue to synthesize acetoin as compared with the control group and animals with the intensified lipogenesis without thiamine administration.     

Metabolic engineering strategies for acetoin and 2,3-butanediol production: advances and prospects

Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and 2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology.     

Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation

Acetoin (3-hydroxy-2-butanone), a very popular food spice is now used in many industries (pharmaceuticals, chemicals, paint, etc.). In this study, an acetoin high producing strain, numbered as JNA-310, was newly isolated and identified as Bacillus subtilis which is safe on food industry, based on its physiological, biological tests and 16S rDNA sequence analysis. When glucose was used as carbon source in fermentation, the fermentation characterizations of this strain were analyzed, and a new phenomenon of reverse transforming 2,3-butanediol which was synthesized from glucose in the fermentation broth to acetoin was detected. Before 96 h, glucose which was mainly transformed to 2,3-butanediol and acetoin was totally consumed, and the yield of the two products were 41.7 and 21.0 g/l respectively. Acetoin was only a by product in the fermentation broth at prophase of fermentation. At the end of fermentation, the yield of acetoin was greatly improved and the yield of 2,3-butanediol was declined and the yield of them were about 42.2 and 15.8 g/l, respectively. The results indicated that 2,3-butanediol was reversely transformed to acetoin.     

Pyruvate degradation via pyruvate formate-lyase (EC 2.3.1.54) and the enzymes of formate fermentation in the green alga Chlorogonium elongatum

Formate was formed in extracts of Chlorogonium elongatum via direct cleavage of pyruvate by a pyruvate formate-lyase (PFL, EC 2.3.1.54). The conversion of PFL to the catalytically active form required S-adenosylmethionine, ferric (2+), photoreduced deazariboflavin as reductant, pyruvate as allosteric effector and strict anaerobic conditions. At the optimum pH (pH 8.0), PFL catalyzed formate formation, pyruvate synthesis and the isotope exchange from [¹⁴C]formate into pyruvate with rates of 30.0, 1.5 and 1.2 nmol min^-1 mg^-1 protein, respectively. Treatment of the active enzyme with O₂ irreversibly inactivated PFL activity (half-time 2 min). In addition to PFL, the activities of phosphotransacetylase (EC 2.3.1.8), acetate kinase (EC 2.7.2.1), aldehyde dehydrogenase (CoA acetylating, EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1) were also detected in extracts of C. elongatum. The occurrence of these enzymes indicates pyruvate degradation via a formate-fermentation pathway during anaerobiosis of algal cells in the dark.Abbreviations DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine+ethane sulfonic acid - PFL pyruvate formate-lyase     

Formation and utilization of acetoin, an unusual product of pyruvate metabolism by Ehrlich and AS30-D tumor mitochondria

[14C]Pyruvate was rapidly non-oxidatively decarboxylated by Ehrlich tumor mitochondria at a rate of 40 nmol/min/mg of protein in the presence or absence of ADP. A search for decarboxylation products led to significant amounts of acetoin formed when Ehrlich tumor mitochondria were incubated with 1 mM [14C] pyruvate in the presence of ATP. Added acetoin to aerobic tumor mitochondria was rapidly utilized in the presence of ATP at a rate of 65 nmol/min/mg of protein. Citrate has been found as a product of acetoin utilization and was exported from the tumor mitochondria. Acetoin has been found in the ascitic liquid of Ehrlich and AS30-D tumor-bearing animals. These unusual reactions were not observed in control rat liver mitochondria.     

Preparation of radioactive diacetyl,acetoin, and 2,3-butylene glycol

Toluene-treated cells of Streptococcus diacetilactis produced large amounts of diacetyl and acetoin without 2,3-butylene glycol. With Na-[3-¹⁴C]pyruvate added to reaction mixtures in place of unlabeled pyruvate, diacetyl with specific activity of 6.1 × 10⁴ cpm/μmol and acetoin with specific activity of 6.8 × 10⁴ cpm/μmol were harvested. Growing cells of Enterobacter aerogens incubated 48 h at 30°C in a complex medium produced large amounts of 2,3-butylene glycol without acetoin or diacetyl. With uniformly labeled [¹⁴C]glucose added to the medium in place of unlabeled glucose, 2,3-butylene glycol with specific activity of 10.8 × 10⁴ cpm/μmol was harvested. The radioactive chemicals were tested and found to be chromatographically homogeneous. Storage frozen in capped containers was especially important for diacetyl, which was found to evaporate rapidly from capped containers at room temperature.     

The precursors of the xylene ring in riboflavine

1. The nature of the precursors of the xylene ring in riboflavine was reinvestigated with growing as well as resting cells of Eremothecium ashbyii. 2. The incorporation of acetoin into riboflavine was very low; further, [2-(14)C]pyruvate and [1-(14)C]acetate were equally effective as precursors of lumichrome, and pyruvate was much more active as a precursor of acetoin. These results exclude acetoin as a direct precursor of riboflavine. 3. Addition of unlabelled glucose decreased the incorporation of [(14)C]acetate into riboflavine more than it decreased the conversion of acetate into carbon dioxide, indicating that acetate is not a direct riboflavine precursor. 4. The incorporation of various sugars and dilution experiments suggest that a derivative of the intermediates of the pentose phosphate cycle is the precursor of the xylene ring in riboflavine.     

Incorporation of Radioactive Acetate into Diacetyl by Streptococcus diacetilactis

Streptococcus diacetilactis was grown in a partially defined, lipoic acid-free medium containing radioactive acetate with and without addition of 0.1% unlabeled sodium pyruvate. Labeled carbon was incorporated into diacetyl, but neither the amount of diacetyl produced nor its specific activity was influenced by addition of pyruvate. Acetoin had low specific activity, indicating that it was a mixture of radioactive and nonradioactive acetoin. The specific activity of acetoin was lower when pyruvate, a precursor of unlabeled acetoin, was added to the medium, which indicated that the radioactive acetoin was produced from radioactive diacetyl by diacetyl reductase. Results substantiate condensation of acetyl-coenzyme A with hydroxyethylthiamine pyrophosphate as the in vivo mechanism for synthesis of diacetyl.     

Enzyme Activities Affecting End Product Distribution by Lactobacillus plantarum in Response to Changes in pH and O(2)

Lactobacillus plantarum catabolic end products changed in response to environmental conditions. While lactate was always the major end product, acetate was produced in alkaline and aerobic environments. Acetoin levels decreased under alkaline conditions. Changes in acetoin dehydrogenase, acetate kinase, NADH oxidase, pyruvate oxidase, and acetate kinase activities correlated with changes in end product distribution.     

Untersuchungen über die L-Äpfelsäure-abbauenden Enzyme von Schizosaccharomyces acidodevoratus

Zusammenfassung Der Abbau von L-Äpfelsäure wird bei Schizosaccharomyces acidodevoratus von einer Malat-Dehydrogenase (EC 1.1.1.37) und einer Oxalacetat-Decarboxylase (EC 4.1.1.3) katalysiert. Stoffwechselprodukt ist Brenztraubensäure. Eine Pyruvat-Decarboxylase (EC 4.1.1.1) und Alkohol-Dehydrogenase (EC 1.1.1.1) wandeln die Brenztraubensäure in Alkohol und CO₂ um. In einer Nebenreaktion entsteht Acetoin. Lactat-Dehydrogenase (EC 1.1.1.27) ist nicht nachweisbar.

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Studies on the Enzymes of Schizosaccharomyces acidodevoratus, decomposing L-malic acid

Summary The decomposition of L-malic acid by Schizosaccharomyces acidodevoratus is catalized by a malate dehydrogenase (EC 1.1.1.37) and an oxaloacetate decarboxylase (EC 4.1.1.3). Pyruvic acid is an intermediate. This acid is converted into ethylalcohol and CO₂ by a pyruvate decarboxylase (EC 4.1.1.1) and an ethylalcohol dehydrogenase (EC 1.1.1.1). The decarboxylation of pyruvic acid yields small amounts of acetoin. The occurrence of lactate dehydrogenase (EC 1.1.1.27) could not be established in our preparations.

     

Influence of temperature on flavour compound production from citrate by Lactobacillus rhamnosus ATCC 7469

The citrate utilization by Lactobacillus rhamnosus ATCC 7469 was found to be temperature-dependent. The maximum citrate utilization and incorporation of [1,5-14C]citrate rate were observed at 37 degreesC. At this temperature, maximum citrate lyase activity and specific diacetyl and acetoin production (Y(DA%)) were observed. The high levels of alpha-acetolactate synthase and low levels of diacetyl reductase, acetoin reductase and L-lactate dehydrogenase found at 37 degreesC led to an accumulation of diacetyl and acetoin. Optimum lactic acid production was observed at 45 degreesC, according to the high lactate dehydrogenase activity. The NADH oxidase activity increased with increasing culture temperature from 22 degreesC to 37 degreesC. Thus there are greater quantities of pyruvate available for the production of alpha-acetolactate, diacetyl and aceotin, and less diacetyl and acetoin are reduced.     

Enzyme Activities Affecting End Product Distribution by Lactobacillus plantarum in Response to Changes in pH and O2

Lactobacillus plantarum catabolic end products changed in response to environmental conditions. While lactate was always the major end product, acetate was produced in alkaline and aerobic environments. Acetoin levels decreased under alkaline conditions. Changes in acetoin dehydrogenase, acetate kinase, NADH oxidase, pyruvate oxidase, and acetate kinase activities correlated with changes in end product distribution.