Effect of acetate on growth and ammonium uptake in the microalga Scenedesmus obliquus

The effect of acetate on growth and rate of ammonium uptake in Scenedesmus obliquus (UTEX 78) was investigated under light-limiting conditions. Addition of acetate to autotrophic cells with a growth constant of 0.71 day⁻¹ resulted in an increase in the growth rate (mixotrophy, k = 1.3 day⁻¹), and in the presence of acetate, growth occurred in the dark (heterophy, k = 0.44 day⁻¹). The rate of ammonium uptake in autotrophy (17.8 amol cell⁻¹ min⁻¹) was similar to that in heterotrophy (17.4 amol cell⁻¹ min⁻¹) but was 3.7 times lower than that in mixotrophy (65.9 amol cell⁻¹ min⁻¹). In general, mixotrophic cells showed optimum ammonium uptake at the acetate concentration at which they were grown. In autotrophy, uptake of ammonium leveled off at about 12.5 μ M while no saturation was observed in mixotrophic cells. An increase in the rate of uptake of ammonium was observed in autotrophic cells within 1 h after the addition of acetate. The activity of isocitrate lyase (EC 4.1.3.1), a key enzyme for the regulation of the glyoxylate cycle responsible for acetate catabolism, showed a 3.9-fold increase in activity after 24 h in the dark in the presence of acetate. The level of isocitrate lyase activity in cells grown for 24 h in the dark in the presence of 0–20 m M acetate also increased as a function of acetate concentration.     

Evidence for the existence of isocitrate lyase activity in methylotrophic Hyphomicrobium strains

Abstract Isocitrate lyase activities were detected in a range of 0.096–0.212 units mg⁻¹ in cell-free extracts of all tested Hyphomicrobium strains grown on methanol as a sole carbon source, although the activities were rapidly lost during storage at 4 °C. When cell-free extracts were incubated with dithiothreitol, after storage the recovery of activity was observed, indicating the involvement of a labile sulfhydryl group in the enzyme. This confirmed the distribution of unstable isocitrate lyase in the genus Hyphomicrobium , and, contrary to previous observations, the operation of the ic ⁺-serine pathway was suggested for the assimilation of one-carbon compounds.     

Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan

A new moderately halophilic Micrococcus sp. 4, isolated from salt-pan water from India, produced extracellular amylase when cultivated aerobically in medium containing wheat bran, peptone, beef extract and sodium chloride. Other salts, such as sodium nitrate, potassium nitrate and sodium sulphate, were also found to be suitable for growth and enzyme production. Maximum amylase activity (1.2 IU ml^-1) was secreted in the presence of 1 mol 1^-1 sodium chloride. The enzyme requires the presence of either sodium chloride, potassium chloride, sodium nitrate, sodium citrate or sodium acetate for its activity. Maximum activity was found in the presence of 1 mol 1^-1 sodium chloride. The pH and temperature optima for enzyme activity were 7.5 and 50°C, respectively.     

The Interaction of Dithiothreitol and Acetyl Coenzyme A in a Radiochemical Assay for Rat Brain ATP:Citrate Oxaloacetate Lyase

Abstract: [¹⁴C]Acetyl-CoA was found to react spontaneously with dithiothreitol to give a relatively apolar product which was readily extractable into a butanol-toluene scintillant. This technique was used in a rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5-¹⁴C]citrate as substrate. The tissue extract, a 14,000 g supernatant, exhibited a lyase activity of approximately 7 nmol acetyl-CoA produced/min per mg supernatant protein, and was inhibited ≥79% by α-ketoglutaric acid (10 m m ), Cu²⁺ (1 m m )and Zn²⁺(1 m m ). [¹⁴C]Oxaloacetate, [¹⁴C]malate and endogenous citrate synthase were found not to interfere significantly with lyase estimations, but NADH was required in the reaction mixture to inhibit acetyl-CoA hydrolase activity.     

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF DMSP LYASE (DIMETHYLPROPIOTHETIN DETHIOMETHYLASE (4.4.1.3)) FROM THE RED ALGA POLYSIPHONIA PANICULATA1

Polysiphonia paniculata Montagne is an intertidal red alga known to produce large amounts of the compound dimethylsulfoniopropionate (DMSP). Conversion of this substrate into dimethylsulfide is accomplished in P, paniculata by an enzyme called DMSP lyase (dimethylpropiothetin dethiomethyla.se (4.4.1.3)). DMSP lyase has been purified and characterized from P. paniculata. Enzymie activity is found in two different proteins: the larger with a molecular weight of 9.26 ± 10⁴ daltons and the smaller with a molecular weight of 3.65 ± 10⁴ daltons. Specific activity of the enzyme is 526 μmols min⁻¹mg⁻¹ for the smaller protein a nd 263 μmols min ⁻¹ mg⁻¹ for the la rger protein. The Michaelis-Menten constant (K_m) is 72.8 μM ± 17.15 and the v_max is 1.62 μmols min⁻¹± 0.928 for the 92.6-kDa protein. The p1 of the larger protein is 5.8 and 5.9 for the smaller protein. Interaction with cysteine protease inhibitors L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane, dithiobis-(2-nitrobenzoate), or N -ethylmaleimide inactivated enzyme activity. The presence of either magnesium or calcium with DMSP lyase enhanced activity al concentrations between 20 and 40 μM but had little effect above these levels. Addition of the divalent chelators ethylenebis(oxyethylenenitrilo) tetraacetic acid and ethylenediaminetetraacetate decreased activity of the enzyme, but activity was restored when either chelator was removed and magnesium or calcium was added to the enzyme .     

Pectinase Production by Bacteria Associated with Improved Preservative Permeability in Sitka Spruce: Synthesis and Secretion of Polygalacturonate Lyase by Cytophaga johnsonii

Cytophaga johnsonii synthesized a polygalacturonate lyase which produced random cleavage of galacturonic acid polymers. No pectin methyl-esterase or hydrolytic pectinase activities could be detected in cultures of the organism. Polygalacturonate lyase synthesis was inducible and also subject to repression by glucose and other compounds. Galacturonic acid was the most effective inducer; lower activities were obtained with citrus pectin, polygalacturonic and polypectic acids. Glucose repression of lyase synthesis was not alleviated by 5 mM-adenosine-3'.5'-cyclic-monophosphate. Enzyme production was growth-linked and ceased when batch cultures entered the stationary phase. In steady-state chemostat cultures lyase activity was maximal at a dilution rate ( D ) of 0.19 h^-1. Polygalacturonate lyase was both cell-bound and free in the supernatant medium. The proportion of free enzyme increased throughout the batch growth cycle and in chemostat cultures over 70% of the activity was cell free at dilution rates below 0.05 h^-1.     

An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Characterization of NADP+-isocitrate dehydrogenase from the host plant cytosol of lucerne (Medicago sativa) root nodules

NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) was purified more than 1500-fold from the host-plant cytosol of Medicago sativa L. cv. Saranac root nodules by ion exchange and affinity chromatography. The forward reaction was characterized. The enzyme exhibited an absolute requirement for a divalent cation (preferably Mn²⁺), had a broad activity optimum from pH 7.5 to 9.0, and was most stable at pH 7.5. The apparent Arrhenius energy of activation was 70.7 kJ mol⁻¹ (20 to 30°C) indicating that the reaction rate of the enzyme was relatively sensitive to temperature. The K_m for isocitrate was 20 μ M and for NADP⁺ 10.7 μ M . Initial velocity and end product inhibition studies of the forward reaction indicate a random bi ter mechanism. End product studies indicated that NADPH was a competitive inhibitor and α-ketoglutarate was a non-competitive inhibitor with respect to isocitrate and NADP⁺. Citrate was a competitive inhibitor with respect to isocitrate. Glutamine was identified as a positive effector when assays were conducted at non-saturating isocitrate concentrations. The potential significance of glutamine regulation of α-ketoglutarate production in a dinitrogen-fixing tissue is discussed.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.     

PURIFICATION AND CHARACTERIZATION OF OX BRAIN NAD+ -DEPENDENT ISOCITRATE DEHYDROGENASE

Abstract— The NAD⁺ -dependent isocitrate dehydrogenase from ox brain has been purified about 130-fold by a method involving affinity chromatography on an NAD⁺ -derivative of agarose. The enzyme preparation is not homogeneous but it is free from contaminating enzyme activities that could interfere with kinetic studies. The kinetic properties of the enzyme did not appear to have been altered by the purification procedure involved. The initial velocity of the reaction showed a sigmoid dependence on the concentration of isocitrate, and ADP behaved as an allosteric activator. The kinetics with NAD⁺ as the substrate were hyperbolic. The molecular weight of the purified enzyme was found to be 285,000 ± 25,000.     

Pectinolytic enzymes of anaerobic fungi

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii , A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100–900 and 10–450 μg galacturonic acid h^-1 mg protein^-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).     

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury-resistant Azotobacter strains

Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1^-1 HgCl₂. Nitrogen-fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1^-1 HgCl₂. Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol 1^-1 HgCl₂. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1^-1 HgCl₂, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N₂-fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.     

Cyanide controls enzymes involved in lipid and sugar catabolism in dormant apple embryos during culture

The activities of alkaline lipase (EC 3.1.1.3, AlkL), isocitrate lyase (EC 4.1.3.1. ICL), pyruvate kinase (EC 2.7.1.40. PK) and glucose–6–phosphate dehydrogenase (EC 1.1.1.49, G6PDH) were determined in cultured, dormant embryos of apple (Malus domestica Borb. cv. Antonówka), pretreated with gaseous HCN. The C₆/C₁ , ratio was estimated in the same material. The activities of AlkL and ICL were not stimulated by HCN pretreatment until the period of maximum stimulation of germination. The activity of G6PDH was inhibited by cyanide only late during the culture of embryos. Therefore, the changes in these activities are considered to be the result and not the cause of enhanced germination. On the other hand, also PK, active very early during the culture of embryos, was modified as a result of the treatment. The cyanide-induced changes in activity of this enzyme in cotyledons (inhibition followed by stimulation) were similar to those in the whole embryo, whereas its changes in the embryonal axis (stimulation followed by inhibition) resembled CN-induced changes in PK in axes of apple seeds submitted to cold stratification (Bogatek and Lewak 1988). The estimation of C₆/C₁ ratios partly confirmed these observations. A role of HCN-induced modifications of PK activity in embryonal dormancy is proposed.     

Methylxanthines as inducers of pectin lyase in Penicillium griseoroseum cultured on sucrose

Pectin lyase (PL) from Penicillium griseoroseum can be induced by xanthine, theobromine, theophylline and especially by caffeine and hypoxanthine (5 mmol l⁻¹ with 0·01% yeast extract (YE)). For caffeine and hypoxanthine, PL activity was, respectively, 5·2 and 3·7 times higher than with YE alone. The simultaneous addition of caffeine or hypoxanthine (5 mmol l⁻¹) and YE (0·1%) had a synergistic effect on PL activity as compared to the addition of these substances alone (0·2% YE; 10 mmol l⁻¹ caffeine; 10 mmol l⁻¹ hypoxanthine). Increasing caffeine concentrations (0–10 mmol l⁻¹) for a constant YE content of 0·01%, resulted in an increase in PL activity and a decrease in mycelial mass. For a constant caffeine concentration (5 mmol l⁻¹) and increasing YE contents (0–0·2%), a higher PL activity and mycelial mass were detected. The addition of caffeine (10 mmol l⁻¹) at the beginning of incubation increased PL activity and decreased mycelial mass, while caffeine added after 12 and 24 h resulted in decreases in PL activity and increases in mycelial mass. The results presented here indicate that methylxanthines, especially caffeine, can induce PL in P. griseoroseum .     

Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity

Samples from biogas digesters, sewage ponds, animal house effluents and food processing wastes were used in enrichment systems seeking anaerobic bacteria producing pectinases. Among the 46 anaerobic consortia developed from various samples, four showed high pectinase activity under static anaerobic conditions. Investigation of fermentation variables showed the optimum conditions for pectinase activity were pH 7.0, 45°C and 72 h of growth with 0.5% pectin in the cultivation medium. A 1.4- to 1.6-fold increase in the pectinase activity was achieved under these conditions. The maximum yield of enzymes (62.72 U ml-1 of pectinase, 4.74 U ml^-1 of polygalacturonase, 113.30 U ml^-1 of pectin lyase, 2.10 U ml^-1 of pectinesterase, 0.75 U ml^-1 of total cellulase and 9.27 U ml^-1 of xylanase) was recorded with the consortia C-S2 developed from decomposed plant samples collected from a pond.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Denitrification and nitrate-reducing bacterial populations in abandoned and reclaimed minesoils

Abstract Microbial populations, nitrogen mineralization potentials, and denitrification enzyme activities were examined in two abandoned carbolithic minesoils. Numbers and activities of bacteria and fungi were lower in nonamended than in lime and/or fly ash amended sites. Rates of aerobic NO₃⁻ production (3 to 38 μg-N kg⁻¹ h⁻¹) and anaerobic NO₃⁻ reduction to N₂O (5 to 68 μg-N kg⁻¹ h⁻¹) were measured. Organisms capable of N₂O production under anaerobic soil conditions were present in low numbers, and their activity was restricted in part by low soil pH. Nondenitrifying nitrate-reducing bacteria were more diverse and in greater numbers than respiratory denitrifiers and may have been responsible for N₂O production in assays measuring denitrification enzyme activity.     

Some effects of low pH and calcium on the growth and tissue mineral content of yearling brown trout, Salmo trutta

Yearling brown trout, Salmo trutta , were exposed to low mineral content water (nominal concentrations of 20μmol 1⁻¹ magnesium, 7.7 μmol 1⁻¹ potassium, 44 μmol 1⁻¹ sodium) over a pH range of 4.0–5.2 with ambient calcium concentrations of 2.5–60 μmol 1⁻¹. All fish died at pH 4.0 and 4.2 irrespective of ambient calcium concentration and also at pH 4.4 with only 2–3 μmol 1 ⁻¹ calcium (that is calcium-free water except for that leached from the diet or excreted by the fish). Good growth rates were obtained over the remaining treatments which extended down to pH 4.4 with as little as 7 μmol 1⁻¹ calcium. When starved, weight loss was inversely correlated with pH. Effects on plasma chloride, percentage dry weight and calcium, potassium sodium, and phosphorus contents of skin, muscle and bone tissue were also investigated. These demonstrated pH effects on mineral metabolism in starved fish, but no effects were detected in fed fish.     

The embryonic axis controls lipid catabolism in cotyledons of apple seeds during germination

Activities of alkaline lipase (AlkL, EC 3.1.1.3), isocitrate lyase (ICL, EC 4.1.3.1), glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and pyruvate kinase (PK, EC 2.7.1.40) were determined in embryos of apple ( Malus domestica Borb. cv. Antonówka) during culture in darkness or at 12 h photoperiod; in both cases either in the presence of gibberellin A₃ (GA₃) or AMO 1618 (inhibitor of GA synthesis). AlkL and ICL were stimulated by light and GA₃; light stimulation was reversed by AMO. G6PDH and PK were not affected by culture conditions. Almost all the activity of all enzymes was found in the cotyledons; only PK was distributed between axis and cotyledons. GA-like activity was found almost exclusively in the embryo axis. Cultured isolated cotyledons lost their sensitivity to light and AMO, but AlkL and ICL were still stimulated by GA₃. Translocation of GA from axis to cotyledons during the culture of embryos is postulated.