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1.
Yuan-Chi Su Nobumasa Tanaka Kōichi Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(9):685-692
In the previous paper, most of the enzymes of the Embden-Meyerhof-Parnas pathway and glucose-6-phosphate dehydrogenase have been demonstrated to be present in cell-free extracts of Brevibacterium divaricatum, No. 1627. In this paper, the presence of condensing enzyme, aconitase, TPN-linked isocitric dehydrogenase, succinic dehydrogenase, fumarase, DPN-linked malic dehydrogenase, TPN-linked malic enzyme, oxalacetic carboxylase, isocitritase and malate synthetase in cell-free extracts of this bacterium was also demonstrated. From these results it was concluded that a strain of Brevibacterium divaricatum which has been found to contain all of the enzymes of the tricarboxylic acid cycle, would be capable of forming the key enzymes of the glyoxylate bypass as well. It suggests that the accumulation of α-ketoglutarate involves the glyoxylate bypass besides the tricarboxylic acid cycle in this bacterium. 相似文献
2.
Washed cell suspensions ofHyphomicrobium spp. were able to oxidize methanol, formaldehyde and formate. This suggested that enzymes for the oxidation of these compounds were present. The pathway of the oxidation of methanol to carbon dioxide and water has been investigated using cell-free extracts. An ammonium-ion-activated, phenazine methosulphate-linked methanol dehydrogenase was detected. This enzyme has a dual substrate specificity for normal primary alcohols and formaldehyde. It has a high pH optimum for activity of 9.5. The pathway is completed by an NAD-linked formate dehydrogenase. This enzyme is inhibited by low concentrations of potassium cyanide, copper sulphate and hypophosphite. 相似文献
3.
Th. Egli J. P. van Dijken M. Veenhuis W. Harder A. Fiechter 《Archives of microbiology》1980,124(2-3):115-121
The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase. 相似文献
4.
N. Arfman E. M. Watling W. Clement R. J. van Oosterwijk G. E. de Vries W. Harder M. M. Attwood L. Dijkhuizen 《Archives of microbiology》1989,152(3):280-288
The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K
m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP
ribulose monophosphate
- HPS
hexulose-6-phosphate synthase
- HPI
hexulose-6-phosphate isomerase
- MDH
methanol dehydrogenase
- ADH
acohol dehydrogenase
- PQQ
pyrroloquinoline, quinone
- DTT
dithiothreitol
- NBT
nitrobluetetrazolium
- PMS
phenazine methosulphate
- DCPIP
dichlorophenol indophenol 相似文献
5.
J. T. Pronk P. de Bruijn J. P. van Dijken P. Bos J. G. Kuenen 《Archives of microbiology》1990,154(6):576-583
Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C1-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol-1 at a dilution rate of 0.05 h-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C1-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate. 相似文献
6.
Brenda S. Speer Ludmila Chistoserdova Mary E. Lidstrom 《FEMS microbiology letters》1994,121(3):349-355
Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well. 相似文献
7.
J. Baratti R. Couderc C. L. Cooney D. I. C. Wang 《Biotechnology and bioengineering》1978,20(3):333-348
Methanol oxidase produced by the yeast Hansenula polymorpha DL-1 was used for the enzymatic oxidation of methanol to formaldehyde. The kinetics of enzyme and protein release during cell desruption were studied at the laboratory scale with a Braun homogenizer and the pilot plant scale with a Manton–Gaulin homogenizer. Conditions were defined for maximum release and retention of high activity in cell-free extracts. Methanol oxidase was immobilized by adsorption on DEAE-cellulose from enzymes in cell-free extracts or from ammonium sulfate purified purified fractions. The kinetics of formaldehyde formation with both soluble and immobilized enzyme was studied in batch and continuous reactors. 相似文献
8.
ELMER MARTIN MICHAEL W. SIMON FRANK W. SCHAEFER ANTONY J. MUKKADA 《The Journal of eukaryotic microbiology》1976,23(4):600-607
SYNOPSIS. The occurrence and levels of activity of various enzymes of carbohydrate catabolism in culture forms (promastigotes) of 4 human species of Leishmania (L. brasiliensis, L. donovani, L. mexicana, and L. tropica) were compared. These organisms possess enzymes of the Embden-Meyerhof pathway but lack lactate dehydrogenase. No evidence could be found for the production of lactic acid by growing cultures and lactic acid could not be detected either in cell-free preparations or after incubation of cell-free extracts with pyruvate and NADH under appropriate conditions. All 4 species possess α-glycerophosphate dehydrogenase and α-glycerophosphate phosphatase which together could regenerate NAD, thus compensating for the absence of lactate dehydrogenase. The oxidative and nonoxidative reactions of the hexose monophosphate pathway are present in all 4 species. Cell-free extracts have pyruvate dehydrogenase activity which allows the entry of pyruvate into and its subsequent oxidation through the tricarboxylic acid cycle. All enzymes of this cycle, including a thiamine pyrophosphate dependent α-ketoglutarate dehydrogenase are present. Both NAD and NADP-linked malate dehydrogenase activities are present. The isocitrate dehydrogenase is NADP specific. There is an active glutamate dehydrogenase which could compete with α-ketoglutarate dehydrogenase for the common substrate (α-ketoglutarate). Replenishment of C4 acids is accomplished by heterotrophic CO2 fixation catalyzed by pyruvate carboxylase. All 4 species have high levels of NADH oxidase activity. Several enzymes thus far not found in any species of Leishmania have been demonstrated. These are: phosphoglucose isomerase, triose phosphate isomerase, fructose-1, 6-diphosphatase, 3-phosphoglycerate kinase, enolase, α-glycerophosphate dehydrogenase, α-glycerophosphate phosphatase, pyruvate dehydrogenase complex, citrate synthase, aconitase, α-ketoglutarate dehydrogenase, glutamate dehydrogenase, and NADH oxidase. 相似文献
9.
Ulrich Härtel Elke Eckel Jürgen Koch Georg Fuchs Dietmar Linder Wolfgang Buckel 《Archives of microbiology》1993,159(2):174-181
Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of glutaryl-CoA. The green enzymes are homotetramers (m=170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%). 相似文献
10.
Catalase has been partially purified from cell-free extracts of methanol-grown Hansenula polymorpha and its peroxidative properties were studied. It was shown that the enzyme is capable of oxidizing methanol, formaldehyde and formate in the presence of hydrogen peroxide. The physiological significance of these reactions in the transduction of energy from the oxidation of methanol in yeasts is discussed. 相似文献
11.
J. P. Van Dijken G. J. Oostra-Demkes R. Otto W. Harder 《Archives of microbiology》1976,111(1-2):77-83
Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K
mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K
mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K
mof 40 mM) but a relatively high affinity for S-formylglutathione (K
mof 1.1 mM). The K
mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts. 相似文献
12.
Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS
Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods
- TY
Tryptone Yeast Extract broth
- OUL
Oxygen uptake with lactate as substrate
- DCPIP
2,6-Dichlorophenolindophenol
- LDH
Lactic dehydrogenase 相似文献
13.
Specific enzymes of ammonium assimilation were measured in cell-free extracts ofNocardia asteroides grown in a synthetic medium with glutamate as the nitrogen source. Cell-free extracts had active glutamine synthetase (GS) and glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) but glutamate dehydrogenase (GDH) could not be detected in the enzyme preparation. This shows that GS/GOGAT is the major pathway of ammonium assimilation inN. asteroides. 相似文献
14.
Hideaki Yamada Akihiko Nagao Hiroshi Nishise Yoshiki Tani 《Bioscience, biotechnology, and biochemistry》2013,77(9):2325-2331
The distribution of glycerol dehydrogenase activity was studied with cell-free extracts of bacteria, yeasts, molds and actinomycetes. High activity was found in 4 strains of bacteria and in 3 strains of molds. The enzymes of bacteria were dependent on NAD+ and those of molds were dependent on NADP+. An isolated gram-positive bacterium, which showed the high activity, was identified as Cellulomonas sp. NT3060. The total and specific activities were associated with growth of this strain and reached the maximum at the early stationary phase. Significant high level activity was detected in cell-free extracts from glycerol and glucose media. 相似文献
15.
Demonstration of formaldehyde dehydrogenase activity in formaldehyde-resistant Enterobacteriaceae 总被引:1,自引:0,他引:1
Clinical isolates of different Enterobacteriaceae strains and genetically modified variants which were resistant to the disinfectant formaldehyde were investigated. In cell-free extracts of all formaldehyde-resistant strains a glutathione-dependent formaldehyde dehydrogenase activity was demonstrated. In contrast cell extracts from formaldehyde sensitive strains did not show any formaldehyde dehydrogenase activity. The enzymatic degradation of formaldehyde seems to play an important role in formaldehyde resistance. 相似文献
16.
The ratios of the oxidation rates of aldose sugars, determined in cell-free extracts of Acinetobacter calcoaceticus, vary with the strain and growth conditions used. Three distinct forms of glucose dehydrogenase with different substrate specificities, occurring in variable proportions in these extracts, are responsible for this effect. One form is the already known soluble glucose dehydrogenase, the other two forms are complexes containing enzyme and components of the respiratory chain. The proportions in which the enzyme forms are found in the cell-free extract correlate with the oxidative behaviour of whole cells with respect to aldose sugars. It is concluded, therefore, that the enzyme forms are not an artefact of the isolation procedure but that they exist as such in vivo. Since the two complexes can be converted into the soluble enzyme form, aldose dehydrogenase can, probably, be integrated in three different ways into the respiratory chain.The presence of glucose during growth does not stimulate aldose dehydrogenase production. This is not surprising since the enzyme has no function in carbon metabolism, except perhaps in strains growing on pentoses at high pH. Therefore, the physiological role of quinoprotein aldose dehydrogenase in this organism may be primarily in energy generation.Non-standard abbreviations quinoproteins
enzymes containing 2,7,9-tricarboxy-1 H-pyrrolo [2,3f] quinoline-4,5-dione (pyrrolo-quinoline quinone) as the coenzyme 相似文献
17.
The intracellular distribution of glycollate dehydrogenase EC 1.2.1.17 has been investigated in extracts of the cyanobacteria (blue-green algae) Anabaena cylindrica, Nostoc muscorum and Chlorogloea fritschii. Most of the enzyme activity was associated with a chlorophyll-containing cell-free pellet, which also exhibited Photosystem I and II activities. Sucrose density gradient centrifugation of this washed pellet resulted in the formation of a green band within which maximal chlorophyll concentration and enzymic glycollate oxidation coincided. Antiserum raised to this fraction obtained from A. cylindrica inhibited glycollate dehydrogenase and Photosystem II activity. The data indicate that most of the cyanobacterial glycollate dehydrogenase is associated with the thylakoids and thus provide evidence for the dual role of these membranes in photosynthetic and respiratory processes.Abbreviations DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
- DCPIP
2,6-dichlorophenolindophenol
- DPC
diphenylcarbazide 相似文献
18.
Summary All enzymes of the Embden-Meyerhof-Parnas pathway were detected in cell-free extracts ofMonilinia fructicola. Hexokinase activity was dependent on the presence of the fluoride ion. The glyceraldehyde-3-phosphate dehydrogenase reaction lasted only a short time. Extracts contained active glucose-6-phosphate and 6-phosphogluconate dehydrogenases of the hexose-monophosphate shunt. No pyruvic dehydrogenase activity could be detected. 相似文献
19.
Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO2, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant. 相似文献
20.
Summary The effect of methanol on the ability of several strains of Aspergillus to produce citric acid from galactose has been investigated. In the absence of methanol, very little production (less than 1 g/l) was observed. In the presence of methanol (final concentration 1% v/v), however, citric acid production and yeilds were increased considerably. Strong relationships were observed between citric acid production and the activities of the enzymes 2-oxoglutarate dehydrogenase and pyruvate carboxylase in cell-free extracts. During citric acid production, in the presence of methanol, the activity of 2-oxoglutarate dehydrogenase was low and that of pyruvate carboxylase high. In the absence of methanol, where little citric acid was produced, the reverse was true. It is suggested that the presence of methanol may increase the permeability of the cell to citrate, and the cell responds to the diminished intracellular level by increasing production via repression of 2-oxoglutarate dehydrogenase. 相似文献