Purification and characterization of carbonyl reductase from Geotrichum candidum

Geotrichum candidum is well known for the reduction of prochiral ketones to chiral alcohol with high yield and excellent enantioselectivity. Carbonyl reductase from G. candidum was purified by ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatographies. Gel filtration chromatography together with SDS-PAGE revealed this protein to be a dimer of 60 kDa subunits. Maximum enzyme activity was found in acetate buffer at pH 5.4 with t_1/2 of 7.13 h at 30 °C and t_1/2 of 2.8 h at 65 °C. The enzyme was inhibited by p-hydroxymercuribenzoate and hydroxylamine indicating the involvement of thiol and carbonyl groups in the reduction reaction catalyzed by the enzyme. Chelating agents also reduced the enzyme activity indicating the requirement of metal ions as cofactors. The purified carbonyl reductase was found to be highly selective for ketones containing naphthyl ring, whereas aryl or hetero-aryl ketones showed very less or no activity at all.     

Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity

Rat heart ornithine decarboxylase activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase had a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O₂^? on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect oupon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of K_m and a reduction of V_max with respect to control activity. The exposure of rates to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

Effect of different phenols on the nadh-oxidation catalysed by a peroxidase from lupin

Cell wall-bound peroxidase (EC 1.11.1.7) from lupin (Lupinus albus) shows a transition from oxidase to peroxidase activity when it oxidizes NADH. The oxidase phase represents a lag period in the time course of the reaction. This phase is phenol-dependent and responsible for hydrogen peroxide formation. Guaiacol, an assay substrate, and p-coumaric, ferulic and sinapic acids, precursors of the cinnamyl alcohols used in the lignification process affect both the length of lag period and the rate of the peroxidase phase of NADH oxidation. The effect of different phenols on the time course of the reaction is related to the efficacy (V_max/K_m ratio) of the enzyme when it is acting on them as a peroxidese.     

Inhibitory properties of endogenous subunit epsilon in the Escherichia coli F1 ATPase.

Homogeneous ? bound tightly to the purified Escherichia coli ATPase (ECF₁ from which ? had been removed and strongly inhibited its ATPase activity. ECF₁ containing ? had a lower specific activity than ECF₁ missing ?, provided that the ATPase assay was carried out at relatively high concentrations of enzyme. Antiserum specific for the ? subunit stimulated the ATPase, as did diluting the enzyme, apparently by dissociating ?. When the ATPase reaction was started by the addition of enzyme, the rate of ATP hydrolysis increased progressively during the first 3 min until a linear steady-state rate was reached. A prior incubation with ATP abolished the lag period and ADP prevented the ATP effect. ECF₁ missing ? gave a linear rate of ATP hydrolysis without a lag, unless ? was rebound to it before the assay. These results suggest that ECF₁ as purified is in an inhibited state due to the presence of the ? subunit, whose interaction with ECF₁ is governed by an equilibrium binding. ATP appears to convert ECF₁ to a form which more readily binds and releases ?.     

Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Tryptophan 2,3-dioxygenase in the development of the stick insect,Carausius morosus Br

Summary Tryptophan 2,3-dioxygenase was studied in the stick insect,Carausius morosus. During the 6th larvl instar, the activity of the enzyme rises from the 5th molt until the premolt stage and declines prior to the imaginal molt (Fig. 1). In adult stick insects, enzyme activity rises continuously to a level twice as high as in the 6th larval instar (Fig. 2). The molecular weight of tryptophan 2,3-dioxygenase was estimated at 120.000; the optimal pH of the reaction is about 7.5. Substrate concentration for half-maximal reaction rate, S_(0.5) is 0.58·10^–3 M; the binding curve is sigmoid with a Hill coefficient of 1.9 (Fig. 4). The reaction is preceded by a lag period of about 30 min.     

Semiaerobic induction of a nitrate reductase activity in growing cultures of Rhodopseudomonas capsulata E1F1 containing NH 4 + ions as nitrogen source

Rhodopseudomonas capsulata E₁F₁ growing under chemo- or photoorganotrophic conditions shows nitrate reductase activity which:

1. Is not repressed by ammonium ions;
2. Is governed by the partial pressure of oxygen in the gas phase.

Upon induction of nitrate reductase activity under optimum conditions, and also in the presence of amonium ions, subsequent addition of nitrate does not result in nitrite production. The specific activity of the enzyme increases by a factor of 10, when the growth medium is supplemented by the addition of nitrate. The physiological role of this ammonium resistant activity is not known.     

The relationship between the state of adenylylation of glutamine synthetase I and the export of ammonium in free-living,N2-fixing Rhizobium

The relationship between ammonium assimilation and ammonium export has been studied in free-living, N₂-fixing Rhizobium sp. 32H1. After 55 to 67 h of microaerobic growth under a gas phase of 0.2% O₂ – 1.0% CO₂ – 98.8% Ar high levels of nitrogenase were observed concomitant with a slightly adenylylated glutamine synthetase (GSI) and some glutamine synthetase (GSII) activity. However, after growth of 89 h, or longer, GSI became adenylylated and the level of GSII had decreased. When the gas phase was shifted to 0.2% O₂ – 1.0% CO₂ – 98.8% N₂, a lag was observed before ammonium export could be detected in the 55 to 67 h cultures. No lag in ammonium export was observed in the cultures previously grown for 89 h. The onset of ammonium export in the 55 to 67 h cultures was found to correlate with the adenylylation state of GSI. There appeared to be no correlation between the level of GSII and the export of ammonium. Neither an increase in the adenylylation level of GSI nor ammonium export was observed when the 55 to 67 h cultures were maintained under the Ar gas mixture.Abbreviations GOGAT Glutamate synthase - GS glutamine synthetase - BES [N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid] - CTAB cetyltrimethylammonium bromide - MES [2-(N-morpholino)-ethane sulfonic acid]     

Ammonium repression of antibiotic and intracellular proteinase production inPenicillium urticae

In this study the addition of ammonium ions (5–30 mM) toPenicillium urticae shake-flask cultures before, during and after the onset of polyketide biosynthesis was examined in a time-dependent manner for its repressive effect on metabolites and a marker enzyme of the patulin pathway and on the intracellular proteinases that also appear during the non-growth or idiophase. A study of the effect of ammonium ion addition, showed that both secondary enzyme and proteinase appearance were maximally delayed if the addition was made before the normal 7 h period of derepression/induction. If added during this period the effect of ammonium ions was progressively less. A reduction in the extracellular ammonium ion concentration from 30 to 4mM appeared to be required to initiate the derepression/induction process. Adding ammonium ions during the appearance of secondary enzymes caused a rapid decrease in specific activity, about 67% for the patulin pathway enzyme and 12% for proteinase. Nitrogen repression exerts a much stronger effect on the expression of polyketide genes as opposed to proteinase genes. Both patulin pathway enzymes and proteinases are subjected to proteolysis, but the proteinases retain much of their activity, whereas the polyketide biosynthetic enzymes do not.     

Specificity of bacteriolytic enzyme II from a soil amoeba, Hartmannella glebae.

Acetylene reduction activity of intact rice plants was measured in closed assay chambers with plants grown in water culture. Acetylene was added to the liquid medium, and the ethylene formed was measured from both gas and liquid phases. After cutoff of mineral nitrogen supply and inoculation of fresh soil, rice plants grown from the seedling stage in water culture exhibited acetylene reduction activity after a lag period. However, rice plants grown in a paddy field and transferred to water culture were more suitable for N₂ fixation studies because of their higher, less variable acetylene reduction activity. The time course of acetylene reduction was monitored by continuous circulation of gas between the gas phase and the liquid phase, and the result showed an initial 2- or 3-h period of lower activity, followed by increased and almost constant activity up to 24 h. The effects on acetylene reduction activity of aeration, ammonium, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea addition are reported. Ammonium was inhibitive at 0.33 mM, and its depressive effect was alleviated by ammonium uptake by the plants.     

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5′_l-imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-β-S and is potentiated at high ratios of GDP-β-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the N_s subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate N_i subunit.     

Influence of nitrogen source on growth and nitrogenase activity inAzotobacter vinelandii

Growth and nitrogenase activity were studied in cultures ofAzotobacter vinelandii growing with dinitrogen, ammonium sulfate, aspartic acid or yeast extract. Nitrogenase activity was measured by means of the C₂H₂ reduction test.In the presence of ammonium sulfate nitrogenase is completely repressed. After exhaustion of ammonia its activity is restored following a diauxic lag period of 30 min. With aspartic acid nitrogenase activity is partially repressed, and growth yield is higher than in the culture growing with N₂ only. This is due to simultaneous use of dinitrogen and aspartate. Fluctuations of nitrogenase activity occurring during exponential growth and the mechanism of their regulation are discussed.Abbreviations NA nitrogenase activity - BNF Burk's nitrogen free medium     

Allantoinase from nodules of pigeonpea (Cajanus cajan)

Allantoinase was purified about 10-fold from nitrogen fixing root nodules of pigeonpea (Cajanus cajan) using (NH₄)₂S0₄ fractionation and chromatography on Sephadex G-100. The purified preparation showed a specific activity of 1.73 nkat/mg protein, M_r of 125 000, pH optimum between 7.5 and 7.7 and K_m of 13.3 mM. The enzyme was heat stable up to 70dg and metal ions, except Hg²⁺, had no effect on the enzyme activity. The enzyme was inhibited significantly by reducing agents. Amino acids, ammonium, nitrate, potential precursors of allantoin and a number of other intermediate metabolites of ureide biosynthetic pathway had no effect on enzyme activity. It is suggested that allantoinase is unlikely to regulate the production of ureides in the nodule tissue.     

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions

Summary.  The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V_max at glutamine concentration of ≤5 mM, and increased K_m for substrate concentration above 5 mM. The activation of the enzyme by bicarbonate was through an increase in V_max. Ammonium and bicarbonate ions may therefore be important physiological regulators of PDG. It is suggested that phosphate and other polyvalent ions may function by preventing product inhibition of the enzyme through promotion of PDG dimer formation. The dimerized enzyme may have a high affinity for glutamine and reduced sensitivity to inhibition by ammonium ions. Received August 10, 2001 Accepted April 1, 2002 Published online August 30, 2002 Acknowledgement This work was supported by University of Zimbabwe research grant to Dr. B. Masola. Authors' address: Dr. Bubuya Masola, Department of Biochemistry, University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe, E-mail: masolab@yahoo.co.uk     

Purification and characterization of acetate kinase from veillonella alcalescens ATCC 17748.

Acetate kinase was isolated in highly purified form from Veillonella alcalescens through a combination of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, and gel filtration. It had a specific activity about 60-fold that of crude extracts. Purity of the enzyme preparation was estimated to be 90% as judged by polyacrylamide gel electrophoresis. The molecular weights of the enzyme as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration were 43,000 and 66,000, respectively. Succinate was unnecessary for the activity of this enzyme. This result is markedly different from that reported previously (Bowman, C. M. et al., 1976, J. Biol. Chem. 251, 3117–3121). This may, however, be due to the difference in bacterial strains used. The enzyme reaction with propionate was equal to about two-thirds that of its reaction with acetate. Apparent K_m values for ATP, acetate, ADP, and acetylphosphate were about 2, 30,0.3, and 1.2 mm, respectively. Phosphate donors, ATP, and acetylphosphate exhibited cooperativity while phosphate acceptors, ADP, acetate, and propionate did not. The enzyme had a broad pH optimum from 7.2 to 10, and required magnesium ions, whose optimal molar ratio to ATP was 1:1. The activity was inhibited by several SH-inhibitors, but not stimulated by free SH groups.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH₄)₂SO₄ (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH₂PO₄. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.     

ATP Production from Adenine by a Self-coupling Enzymatic Process: High-level Accumulation under Ammonium-limited Conditions

To improve ATP production from adenine, we optimized cultivation and reaction conditions for the ATP producing strain, Corynebacterium ammoniagenes KY13510. In the conventional method, 28% NH₄OH has been used both to adjust pH during cultivation and reaction, and to provide nitrogen for cell growth. In the ATP-producing reaction, high concentrations of inorganic phosphate and magnesium ion are needed, which form magnesium ammonium phosphate (MgNH₄PO₄) precipitate. To keep inorganic phosphate and magnesium ions soluble in the reaction mixture, it was indispensable to add phytic acid as a chelating agent of divalent metal ions. Under such conditions, 37 mg/ml (61.2 mM) ATP was accumulated in 13 h (Appl. Microbiol. Biotechnol. 21, 143 1985). If ammonium ion was depleted from the reaction mixture to avoid MgNH₄PO₄ formation, we expected that there was no need to add phytic acid and ATP accumulation might be improved. Therefore, we obtained the cultured broth of C. ammoniagenes KY13510 strain with low ammonium ion content (less than 1 mg/ml as NH₃) by the method that a part of alkali solution (28% NH₄OH) for pH control was replaced with 10 N KOH. Using this culture broth, ATP producing reaction was done in 2-liter jar fermentor, controlling the pH of the reaction mixture with 10 N KOH. Under these conditions, the rate of ATP accumulation improved greatly, and 70.6 mg/ml (117 mM) ATP was accumulated in 28 h. The molar conversion ratio from adenine to ATP was about 82%. Phytic acid was slightly inhibitory to ATP formation under these ammonium-limited conditions.     

Purification and properties of invertase from a glutamate-producing bacterium

Invertase was purified from the cell extracts of the glutamic acid bacterium (Brevibacterium divaricatum) by ammonium sulfate fractionation, batch theatment with DEAE-cellulose, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was proved to be almost homogeneous by polyacrylamide gel electrophoresis.The molecular weight of the enzyme was estimated to be 92,000 by both gel filtration and SDS-polyacrylamide gel electrophoresis methods. The optimum pH and temperature for the activity were 6.8 and 40°C. The enzyme was highly specific to sucrose as substrate, having only 10% as much activity toward raffinose as that toward sucrose, and being inert toward other disaccharides: maltose, trehalose, lactose, melibiose and cellobiose. The K_m value for sucrose was 0.19 M. The enzyme required phosphate or arsenate ions for activity. Monovalent or divalent Cu ions and sulfhydryl reagents inhibited the enzyme.