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At least 19 recordsLinked to original sources
PubMed1976

Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism.

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h-1 showed a maximal capacity to oxidize excess methanol (QmaxO2) which was 1.6 times higher than the rate required to sustain the growth rate (QO2). When the dilution rate was decreased to 0.03 h-1, QmaxO2 of cells increased to a value of more than 20 times that of QO2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent Km for methanol of 1.3 mM in air saturated reaction mixtures and the apparent Km of the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM. The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymprpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells X h.

Alcohol Oxidoreductases↗
PubMed1978

Microbial oxidation of methane and methanol: crystallization of methanol dehydrogenase and properties of holo- and apomethanol dehydrogenase from Methylomonas methanica.

Procedures are described for the purification and crystallization of methanol dehydrogenase from the soluble fraction of the type I obligate methylotroph Methylomonas methanica strain S1. The crystallized enzyme is homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme had a high pH optimum (9.5) and required ammonium salt as an activator. In the presence of phenazine methosulfate as an electron acceptor, the enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight as well as subunit size of methanol dehydrogenase was 60,000, indicating that it is monomeric. The sedimentation constant (s(20,w)) was 3.1S. The amino acid composition of the crystallized enzyme is also presented. Antisera prepared against the crystalline enzyme were nonspecific; they cross-reacted with and inhibited the isofunctional enzyme from other obligate methylotrophic bacteria. The crystalline methanol dehydrogenase had an absorption peak at 350 nm in the visible region and weak fluorescence peaks at 440 and 470 nm due to the presence of a pteridine derivative as the prosthetic group. A procedure was developed for the preparation of apo-methanol dehydrogenase. The molecular weights, sedimentation constants, electrophoretic mobilities, and immunological properties of apo- and holo-methanol dehydrogenases are identical. Apo-methanol dehydrogenase lacked the absorption peak at 350 nm and the fluorescence peaks at 440 and 470 nm and was catalytically inactive. All attempts to reconstitute an active enzyme from apo-methanol dehydrogenase, using various pteridine derivatives, were unsuccessful.

Alcohol Oxidoreductases↗
PubMed1976

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha.

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes D-aminoacid oxidase and L-alpha-hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.

Alcohol Oxidoreductases↗
PubMed1975

Oxidation of C1 compounds by particulate fractions from Methylococcus capsulatus: properties of methanol oxidase and methanol dehydrogenase.

Methanol (and formaldehyde) oxidizing activities in crude extracts of Methylococcus capsulatus are associated mainly with particulate fractions sedimenting between 3,000 and 40,000 X g. Most of the phenazine methosulfate (PMS)-dependent methanol (and formaldehyde) dehydrogenase activity observed resides in the soluble fraction but represents only 40% of the total (PMS dependent plus independent) activity. Both PMS-dependent methanol dehydrogenase activity and PMS-independent methanol oxidase activity are found in particulate fractions, and the PMS-dependent dehydrogenase is easily solubilized by treatment with certain phospholipases or detergents. The properties of the PMS-dependent dehydrogenase activities in the soluble fraction and that solubilized from the particles suggested that they may be identical proteins. Their pH optima, temperature dependence, thermolabilities, and sensitivities to the presence of specific antisera were indistinguishable. Homogeneous preparations of the enzyme proteins obtained from the soluble fractions of extracts and the particulate fractions solubilized by detergents had similar: (i) electrophoretic mobilities in native and denatured states (subunit size in sodium dodecyl sulfate 62,000 daltons); (ii) molecular radii under native conditions, (iii) visible absorption spectra, lambdamax 350 nm, (iv) kinetic constants for methanol and formaldehyde; (v) substrate specificity; and (vi) immunological characteristics--antisera to each enzyme preparation showed precipitin lines of identity to either of the enzymes. It is suggested that the major site of methanol and formaldehyde oxidation in M. capsulatus occurs on the intracytoplasmic membranes in vivo and is coupled to oxygen reduction.

Alcohol Oxidoreductases↗
PubMed1976

Microbial oxidation of methane and methanol: crystallization and properties of methanol dehydrogenase from Methylosinus sporium.

Obligate methylotrophs are divisible into two types on the basis of ultrastructural biochemical characteristics. Both groups possess a soluble phenazine methosulfate (PMS)-dependent methanol dehydrogenase. In addition, particulate PMS-dependent methanol dehydrogenase and PMS-independent methanol oxidase have been found in the type I membrane group. A procedure was developed for the crystallization of methanol dehydrogenase from the soluble fraction of the type II obligate methylotroph Methylosinus sporium. This is the first report of a crystalline methanol dehydrogenase from a methylotrophic bacterium. The crystallized enzyme is homogeneous as judged by ultracentrifugation and by acrylamide gel electrophoresis. In the presence of an electron acceptor (phenazine or phenazinium compound) and an activator (ammonium compound), the crystallized enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight of the enzyme as estimated by gel filtration is approximately 60,000, and as estimated by sedimentation equilibrium analysis it is 62,000. The sedimentation constant (S20,W) is 2.9. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 60,000. The amino acid composition and spectral properties of the enzyme are also presented. Antisera prepared against the crystalline enzyme are nonspecific, they cross-reacted and inhibited isofunctional enzymes from other obligate methylotrophic bacteria.

Alcohol Oxidoreductases↗
PubMed1976

Oxidation of methanol, formaldehyde and formic acid by methanol-utilizing yeast.

Methanol-utilizing yeast, Candida boidini 11 Bh, characterized by high tolerance to methanol during growth, displays even higher tolerance when the oxidation rate by intact cells is tested. Low respiration activity is found even at 22% v/v of methanol. The half-saturation constant was 17-18 mM. The half-saturation constants for the two oxidation intermediates, formaldehyde and formic acid were 3.5-4.0 an .d 30-33 mM, respectively. When applied together with standard concentration of methanol, very low concentrations of both intermediates stimulated the oxidation rate. These results are discussed in connection with the relationship between growth and oxidation, the tolerance to high concentrations of inhibitory products and the mechanism of inhibition.

Candida↗
PubMed1975

Pyridine nucleotide-linked oxidation of methanol in methanol-assimilating yeasts.

An alcohol dehydrogenase linked to nicotinamide adenine dinucleotide and requiring glutathione has been isolated and partially purified from two methanol-assimilating yeasts. It differs from previously described methanol-oxidizing enzymes in pH optima, electron acceptor specificity, substrate specificity, inhibition pattern, and stability.

Alcohol Oxidoreductases↗
PubMed1975

Comparison of a 9-phenanthrene methanol (WR33063), a 4-quinoline methanol (WR30090), and quinine for falciparum malaria in Thailand.

Quinine was compared with a 9-phenanthrene methanol (WR33063) and a 4-quinoline methanol (WR30090) for the treatment of 207 patients with falciparum malaria in Southeast Thailand. Quinine eradicated parasitaemia (average 70 hours) more rapidly than either WR30090 (72 hours) or WR33063 (77 hours). But WR33063 had a higher cure rate (92%) than WR30090 (86%) or quinine (85%). The mean duration of fever and of parasitaemia were combined with the failure rate to form an arbitrary efficacy index. Using this concept WR33063 was the most effective drug. The recrudescence rate correlated with the degree and duration of parasitaemia and with the duration of fever. WR33063 was the least toxic drug. Side effects associated with WR30090 appeared to be headache, backache and urticaria. Quinine was the most toxic drug. All 3 drugs were inconvenient in having to be administered every 8 hours for 6 days. One patient did not respond to oral quinine but did respond to an intravenous quinine infusion (IVQ). A "Medication Ward Round" was perfected during the study and comprised sequential history, drug administration, physical examination, dose notation and patient observation. Falciparum nephrosis was diagnosed in one patient.

Adolescent↗
PubMed1975

Studies on methanol - oxidizing yeast. III. Enzyme.

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.

Aerobiosis↗
PubMed1978

Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol.

Methanosarcina strain 227 exhibited exponential growth on sodium acetate in the absence of added H(2). Under these conditions, rates of methanogenesis were limited by concentrations of acetate below 0.05 M. One mole of methane was formed per mole of acetate consumed. Additional evidence from radioactive labeling studies indicated that sufficient energy for growth was obtained by the decarboxylation of acetate. Diauxic growth and sequential methanogenesis from methanol followed by acetate occurred in the presence of mixtures of methanol and acetate. Detailed studies showed that methanol-grown cells did not metabolize acetate in the presence of methanol, although acetate-grown cells did metabolize methanol and acetate simultaneously before shifting to methanol. Acetate catabolism appeared to be regulated in response to the presence of better metabolizable substrates such as methanol or H(2)-CO(2) by a mechanism resembling catabolite repression. Inhibition of methanogenesis from acetate by 2-bromoethanesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M. Labeling studies also showed that methanol may lie on the acetate pathway. These results suggested that methanogenesis from acetate, methanol, and H(2)-CO(2) may have some steps in common, as originally proposed by Barker. Studies with various inhibitors, together with molar growth yield data, suggest a role for electron transport mechanisms in energy metabolism during methanogenesis from methanol, acetate, and H(2)-CO(2).

Acetates↗
PubMed1975

Utilization of methanol by rhodospirillaceae.

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophia strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or fomaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.

Bicarbonates↗
PubMed1978

Aerobic and anaerobic growth of Paracoccus denitrificans on methanol.

1. The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate. 2. Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme. 3. Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions. 4. Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured. 5. From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.

Aerobiosis↗
PubMed1979

The effects of methanol on the glutamate dehydrogenase reaction at 0 degrees C.

The effects of 0-30% methanol (vol/vol) on the Km an Vm values for both the forward and reverse directions of the L-glutamate dehydrogenase reaction were determined at 0 degrees C. The decrease in temperature alone had very little effect on these parameters. However, in the forward reaction, 30% methanol resulted in a 14-fold decrease in the Km value for glutamate, a slight decrease in the Km value for NADP, and a thirty-fold decrease in Vm. Substrate inhibition by glutamate was observed at concentrations greater than 4 mM. In the reverse reaction, 30% methanol caused a decrease in the Km values for alpha-ketoglutarate and ammonia and a 10-fold decrease in Vm. Substrate inhibition by both alpha-ketoglutarate and NADPH was observed at concentrations of either substrate above 0.03 mM. The dependence of Km for glutamate and Vm values for the forward reaction on methanol concentration suggests that they are similarly affected by methanol, in direct contrast to results obtained for NADP. Methanol appeared to cause a general tightening of complexes, which may arise from an effect on the "activities" of species in solution. The use of methanol not only allows for the study of reaction intermediates by slowing the reaction with the cryogenic method, but may also serve as a mechanistic probe by affecting several polarity as well as Km, Vm, and K1 values.

Animals↗