Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO₄ immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO₄ and ZnSO₄, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn²⁺ or Mg²⁺. Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe²⁺ to these cells caused a rapid recovery of EFE while Cu²⁺, Zn²⁺ and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10–20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.     

Phosphatidyl inositol-specific phospholipase C from Clostridium novyi type A

From the culture broth of Clostridium novyi type A, phosphatidyl inositol-specific phospholipase C was separated from the major part of phospholipase C (γ-toxin) which hydrolyzes phosphatidyl choline, phosphatidyl ethanolamine, and sphingomyelin. Sodium deoxycholate stimulated the activity of phosphatidyl inositol phospholipase C. The concentration of sodium deoxycholate for maximal stimulation was 0.2% with 2 mm phosphatidyl inositol. Divalent cations (Mg²⁺, Ca²⁺, and Zn²⁺) were rather inhibitory above 10^?3m. Phosphatidyl inositol phospholipase C was not inhibited by EDTA or o-phenanthroline. When phosphatidyl inositol phospholipase C was incubated with rat liver slices, not only alkaline phosphatase but also 5′-nucleotidase was liberated into the soluble fraction.     

Optimization of the hide powder azure assay for quantitating the protease of Pseudomonas fluorescens

The extracellular protease of Pseudomonas fluorescens NC 3 was optimally active at 40°C in a reaction mixture containing: 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 6.6), 0.5 mM CaCl₂, and 25 mg hide powder azure in 5 ml total volume. Divalent cation chelators, i.e., EDTA, o-phenanthroline, citrate or phosphate, inhibited the enzyme. Protease production by P. fluorescens NC 3 was initiated during late-logarithmic-growth phase in a sodium caseinate medium and reached its maximum at the onset of the stationary phase.     

Degradation of some sugars and sugar acids by the nonphosphorylated D-gluconate pathway in Aspergillus ustus

The degradation of fifteen sugars, sugar acids and related substrates were examined using cellfree extracts of Aspergillus ustus growing on D-glucose, D-mannose, D-galactose or D-gluconate as the only carbon source. D-gluconate was superior for the induction of the enzymes capable for the degradation of some of these substrates. The addition of 0.5% malt extract with D-gluconate to the growth medium or the presence of shaking conditions resulted to an increase in the degradation of those substrates, whileas the incorporation of 0.5% malt extract alone to the medium has no effect. Extracts of D-gluconate-grown mycelia of A. ustus degraded D-gluconate > D-galactonate > 1 : 5 gluconolactone and > L-arabonate nonphosphorolytically more effectively. Optimum pH and temperature for the degradation of D-gluconate were found to be 8.0 and 40°C, respectively. Thermal stability studies on the behaviour of D-gluconate dehydratase showed that this enzyme was stable at 50°C and 60°C for 30 and 5 minutes, respectively. Specific activity of this enzyme was increased three times when cell-free extracts were incubated at 60°C for 5 minutes. MgCl₂ and CoSO₄ were good activators, while CaCl₂ p-mercurychlorobenzoate (PMCB), sodium arsenite, ZnSO₄, CuSO₄, iodoacetic acid, MnCl₂ and FeSO₄ were potent inhibitors for D-gluconate dehydratase activity. K_m was calculated for D-gluconate and found to be 2.5 × 10^?2 M.     

Bactericidal and non-cytotoxic activity of bacteriocin produced by Lacticaseibacillus paracasei F9-02 and evaluation of its tolerance to various physico-chemical conditions

This study aims to explore novel lactic acid bacteria (LAB) from breast-fed infants' faeces towards characterizing their antimicrobial compound, bacteriocin. The LAB, Lacticaseibacillus paracasei F9-02 showed strong antimicrobial activity against clinical pathogens. Their proteinaceous nature was confirmed as the activity was completely abolished when treated with proteinaceous enzymes and retained during neutral pH and catalase treatment. The purified bacteriocin showed antimicrobial activity at the minimum inhibitory concentration (MIC) value of 7.56 μg/ml against vancomycin-resistant Enterococcus sp. [vancomycin-resistant enterococcal (VRE)], and methicillin-resistant Staphylococcus aureus (MRSA), 15.13 μg/ml against Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhi and 30.25 μg/ml against Shigella flexneri. Present study also proved the bactericidal, non-cytotoxic and non-hemolytic nature of bacteriocin. Additionally, bacteriocin retained their stability under various physico-chemical conditions, broad range of pH (2–10), temperature (40–121°C), enzymes (amylase, lipase and lysozyme), surfactants [Tween-20, 80, 100 and sodium dodecyl sulfate (SDS)], metal ions (CaCl₂, FeSO₄, ZnSO₄, MgSO₄, MnSO₄, CuCl₂) and NaCl (2%–8%). The molecular weight of bacteriocin (~28 kDa) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), functional and active groups were assessed by Fourier Transform-Infrared (FT-IR). To our knowledge, this is the first study reporting L. paracasei from breast-fed infants' faeces and assessing their antimicrobial compound, bacteriocin. The study results furnish the essential features to confirm the therapeutic potential of L. paracasei F9-02 bacteriocin.     

Production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae

The production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae from sheep were studied. D. flagrans was grown in liquid medium with glucose, casein, bibasic potassium phosphate (K₂HPO₄), magnesium sulfate (MgSO₄), zinc sulfate (ZnSO₄), ferrous sulfate (FeSO₄), and copper sulfate (CuSO₄). The proteolytic activity was measured within varied pHs and temperatures. To determine the thermostability, the crude extract was incubated at 28°C for 72 h. To study the effect of different chemical compounds on the activity of the crude extract, the samples were incubated in solutions containing (10 mM): calcium chloride (CaCl₂), copper II sulfate (CuSO₄), zinc sulfate (ZnSO₄), magnesium sulfate (MgSO₄), inhibitor phenylmethylsulfonyl fluoride (PMSF), and 0.5% SDS. Results showed that the highest activity obtained (79.23 U/mL) was at pH 9.0, while the optimum temperature was 60°C (119.6 U/mL). The thermostability analysis demonstrated that after 72 h the activity was maintained or increased. It was found that the CuSO₄, ZnSO₄, and PMSF strongly inhibited the proteolytic activity. Moreover, the MgSO4 and SDS, caused a weak inhibition of the proteolytic activity. There was a significant (P<0.01) reduction in number of treated L₃ when compared to control (94.2%). The results suggest that the crude extract produced by D. flagrans (AC001) in liquid medium exerted larvicidal activity on trichostrongilid L₃ and therefore may contribute to a large-scale industrial production.     

An in vitro study of metal ion-induced lipid peroxidation in giant fresh water prawn Macrobrachium rosenbergii (de MAN)

Though metal ions are essential components of many cellular functions, their overexposure to organisms lead to oxidative stress through the formation of reactive oxygen species (ROS). Lipid peroxidation (LPX) is the oxidative deterioration of membrane lipids and considered as an index of oxidative stress. In the present study in vitro effect of various metals (FeCl₃, FeSO₄, CuSO₄, CdCl₂, and ZnSO₄) on the lipid peroxidation of gills and hepatopancreas of Giant Freshwater prawn, Macrobrachium rosenbergii, was compared with respect to dose and duration. The results clearly indicate that among all the metals investigated, FeCl₃ and CdCl₂ are more potent in inducing LPX, and FeCl₃ is more toxic than FeSO₄ in inducing LPX in the hepatopancreas. ZnSO₄ exhibits a moderate toxicity while CuSO₄ is least toxic and also inhibits LPX at higher concentration. Thus results of the present investigation suggest that all the metal ions investigated in the present study are capable of inducing oxidative stress in gills and hepatopancreas of M. rosenbergii     

Inactivation of photosynthetic oxygen evolution by o-phenanthroline and LiClO4 in Photosystem 2 of the pea

We examined the effects of o-phenanthroline and LiClO₄ on oxygen evolution and electron transport in the Photosystem 2 complex of the pea. Treatment of Photosystem 2 particles with a combination of 3.0 mM o-phenanthroline and 1.0 M LiClO₄ for 30–40 min at 0°C decreased the oxygen-evolving activity with the electron acceptor (either phenyl-p-benzoquinone or 2,6-dichlorophenol indophenol) to less than 5% of the original level. However with the same treatment, the electron-transport activity from an artificial electron donor, 1,5-diphenylcarbohydrazide, to 2,6-dichlorophenol indophenol remained at 60% of the original activity. The amount of manganese in the Photosystem 2 complex decreased in parallel with the loss of oxygen evolution following treatment. These observations suggest that the treatment of the Photosystem 2 complex with o-phenanthroline and LiClO₄ inhibits electron transport on the oxygen-evolving side much more significantly than on the electron-acceptor side.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - DPC 1,5-diphenylcarbo hydrazide - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - PBQ phenyl-p-benzoquinone - PS 2 Photosystem 2     

Uptake of protoporphyrin IX by isolated rat liver mitochondria.

The ability of rat liver zinc-thionein to donate its metal to the apo-enzymes of the zinc enzymes horse liver alcohol dehydrogenase, yeast aldolase, thermolysin, Escherichia coli alkaline phosphatase and bovine erythrocyte carbonic anhydrase was investigated. Zinc-thionein was as good as, or better than, ZnSO₄, Zn(CH₃CO₂)₂ or Zn(NO₃)₂ in donating its zinc to these apo-enzymes. Apo-(alcohol dehydrogenase) could not be reactivated by zinc salts or by zinc-thionein. Incubation of the other apo-enzymes with near-saturating amounts of zinc as ZnSO₄, Zn(CH₃CO₂)₂, Zn(NO₃)₂, or zinc-thionein resulted in reactivation of the apo-enzymes. With apo-aldolase zinc-thionein gave 100% reactivation within 30min. Reactivation by ZnSO₄ and Zn(CH₃CO₂)₂ was complete and instantaneous. Zinc-thionein was somewhat better than Zn(NO₃)₂ in completely reactivating apo-thermolysin. With apo-(alkaline phosphatase) 43% reactivation was obtained with Zn(CH₃CO₂)₂ and 18% with zinc-thionein. With apo-(carbonic anhydrase) zinc-thionein was better than ZnSO₄, Zn(CH₃CO₂)₂ or Zn(NO₃)₂, with a maximal reactivation of 54%. That zinc was really being transferred from zinc-thionein to apo-(carbonic anhydrase) was shown by the fact that 2,6-pyridine dicarboxylic acid and 1,10-phenanthroline had minimal effects on the reactivation of apo-(carbonic anhydrase) when added after the incubation {[apo-(carbonic anhydrase)+zinc thionein]+chelator}, but inhibited reactivation when added before the incubation {apo-(carbonic anhydrase)+[zinc-thionein+chelator]}. These observations support the idea that zinc-thionein can function in zinc homeostasis as a reservoir of zinc, releasing the metal to zinc-requiring metalloenzymes according to need.     

Absorption and transfer of fe and mn in germinating sorghum

The absorption of Fe from FeSO₄, FeEDTA, and FeEDDHA (ferric ethylenediaminedi (o-hydroxyphenylacetate)), and Mn from MnSO₄, MnEDTA, and MnEDDHA, by germinating sorghum (Sorghum vulgarie Pers. var. M 35-1) was studied. The seeds were found to absorb Fe and Mn from all the sources, and these ions moved to the scutellum, shoot, and root. EDDHA facilitated greater translocation of Fe and Mn from the seed to the shoot and root. The translocation of Fe was more towards the root than to the shoot, whereas it was the reverse in the case of Mn.     

Optimized production of Pichia guilliermondii biomass with zinc accumulation by fermentation

Zinc is an essential nutrient that plays an important role in several biological processes of living organisms. When bound to an organic substrate, Zn is more efficiently absorbed by organisms, has a high biological activity and a low toxicity. Due to its ability to incorporate metals, yeast biomass has been used frequently as a delivery vehicle for many mineral supplements. This study describes the screening of strains of yeast for production of biomass enriched with Zn by submerged fermentation. Five strains of yeasts, belonging to the genera Saccharomyces, Kluyveromyces and Pichia, were evaluated. The highest Zn concentration was 6820 mg/kg of dry weight biomass, using Pichia guilliermondii Wickerham LPB 063 after 120 h of cultivation in a medium with 0.5 g/L ZnSO₄. Process conditions were optimized using statistical experimental design methodology. Four parameters were identified in the 2⁸⁻⁴ fractional factorial design as having a significant effect on Zn accumulation: ZnSO₄ and Fe₂(SO₄)₃ concentrations, time of addition of the ZnSO₄ solution and concentration of soybean molasses. In the 3² experimental design, the influence of ZnSO₄ and Fe₂(SO₄)₃ concentrations were studied more closely. The highest Zn concentration (75,090 mg/kg dry weight) in the biomass was reached using the conditions: ZnSO₄, 10.0 g/L; Fe₂(SO₄)₃, 0.1 g/L in Erlenmeyer flasks. A batch liquid fermentation was carried out in a 2 L bioreactor for production of P. guilliermondii Wickerham LPB 063 containing organically bound Zn. The concentration of organically bound Zn after 144 h of fermentation was of 96,030 mg/kg, with a biomass production of 30 g/L. The maximum specific growth rate obtained (μ_max) was 0.0077/h, while the maximum productivity of biomass was at 0.1511 g/L/h.     

Interaction of the high-spin Fe atom in the photosystem II reaction center with the quinones QA and QB in purified oxygen-evolving PS II reaction center complex and in PS II particles

EPR signals in the high-spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen-evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264-275]. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or o-phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o-phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high-spin signals are due to Q₄₀₀, Fe(III) atom interacting with the PS II primary electron acceptor quinone Q_A as reported and that the Fe atom also interacts with the secondary acceptor quinone Q_B. This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o-phenanthroline in a somewhat different manner.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

In vitro shoot growth of Brugmansia × candida Pers

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida ‘Creamsickle’. Several mineral nutrient experiments were conducted to determine the effect of NH₄⁺, NO₃⁻, K⁺, FeSO₄/EDTA, ZnSO₄, MnSO₄, and CuSO₄ on quality, leaf width and length, size and weight of shoot mass, and shoot number. The experiment to determine the levels of NH₄⁺, NO₃⁻, and K⁺, was conducted as a 2-component NH₄⁺: K⁺ mixture crossed by [NO₃⁻] and resulted in an experimental design free of ion confounding and capable of separating the effects of proportion and concentration. The results of the NH₄⁺-K⁺-NO₃⁻ experiment revealed a region in the design space where growth was significantly improved; the region generally had lower total nitrogen and lower NH₄⁺:K⁺ ratios than MS medium. The experiments to determine the appropriate levels of Fe, Zn, Mn, and Cu were conducted at six log levels ranging from 0 to 1 mM. Of the four metal salts tested, MnSO₄ had the least effect on in vitro shoot growth and its concentration was reduced from 0.1 mM (MS level) to 0.001 mM. CuSO₄ had large effects on in vitro shoot growth and was increased from 0.0001 mM to 0.001 mM. A 2-level factorial of NH₄⁺-K⁺-NO₃⁻, FeSO₄/EDTA, and ZnSO₄ was conducted and several formulations identified for their improvements of quality and growth. In addition to the changes to MnSO₄ and CuSO₄, these formulations were characterized by lower levels of NH₄⁺, K⁺, NO₃⁻ and Zn, and higher levels of FeSO₄/EDTA. Overall, several nutrient formulations were identified as superior to MS medium for growth of in vitro shoot cultures of B. ‘Creamsickle’.     

Glucosinolate Biosynthesis: Sulfation of Desulfobenzylglucosinolate by Cell-Free Extracts of Cress (Lepidium sativum L.) Seedlings

After removal of myrosinase activity by concanavalin A-Sepharose 4B chromatography, cell-free extracts of light-grown cress (Lepidium sativum L.) seedlings, catalyzed the sulfation of desulfobenzylglucosinolate (K_m, 0.23 millimolar) to benzylglucosinolate using PAPS (K_m, 1 millimolar) as sulfur donor. Sulfotransferase activity, which was optimal at pH 9.0, was stimulated by MgCl₂, MnCl₂, β-mercaptoethanol, and dithiothreitol and was inhibited by ZnSO₄ and SH-reagents. The enzyme also sulfated desulfoallyglucosinolate to allylglucosinolate (sinigrin) but was inactive towards all phenylpropanoids and flavonoids tested.     

Purification and characterization of cocoonase from the silkworm Bombyx mori

Cocoonase (CCN) which facilitates the degradation of a cocoon is recognized as a trypsin-like serine protease. In this study, CCN from the silkworm Bombyx mori was purified and comprehensively characterized. Its activity was maximal at about pH 9.8. It was stable above pH 3.4 at 4?°C and below 50?°C at pH 7.5. CuSO₄, FeSO₄, and ZnSO₄ showed inhibitory effects on CCN, but other salts improved activity. Typical trypsin inhibitors inhibited CCN, but the relative inhibitory activities were much lower than those against bovine trypsin. An extract of cocoon shells inhibited trypsin, but it was only slightly inhibitory against CCN. There were significant differences in catalytic efficiencies and substrate specificities as between CCN and bovine trypsin.     

Production and regulation of extracellular chitinase from the entomopathogenic fungus Isaria fumosorosea

Extracellular chitinase production by the entomopathogenic fungus, Isaria fumosorosea IF28.2 was studied by using submerged fermentation. Maximum chitinase production (178.34±3.91 mU/mL) was obtained when fermentation was carried out at 25°C for 120 h using 72-h-old mycelium in a medium. The effect of inoculum size on chitinase activity was also observed and maximum chitinase activity (159.41±2.91 mU/mL) was obtained with an inoculum size of 3 discs while an incubation period of 96 h proved the most active inducer of chitinase production yielding a chitinase activity of 186.14±3.81 mU/mL. Colloidal chitin (1.5%, w/v) proved to be the best concentration. The optimum pH for chitinase production was 5.7 while 25°C proved to be the best temperature for chitinase production. Supplementation of additional carbon source like 1.5% N-acetylglucosamine (GlcNAc) showed further enhancement in chitinase production. The divalent metal salts, CaCl₂, MgCl₂ and ZnSO₄, inhibited chitinase activity at 10 and 100 mM concentration, whereas inhibition of chitinase activity by KCl, FeSO₄ and EDTA was observed only at higher concentrations. The results presented in this study increase the knowledge on chitinase production in I. fumosoroseus opening new avenues for the study of the role of this enzyme in virulence against different insect pests during the infection process.     

Optimisation of exopolysaccharide production by Gomphidius rutilus and its antioxidant activities in vitro

The production conditions of the Gomphidius rutilus exopolysaccharides (GREP) in submerged culture were optimised, and the antioxidant activities of GREP in vitro were evaluated. The optimal culture medium constituents were determined as follows: 30 g/L sucrose, 3.0 g/L soybean meal, 0.25 g/L MgSO₄, 1.5 g/L K₂HPO₄, 0.5 g/L KH₂PO₄, 0.03 g/L ZnSO₄, and 0.01 g/L FeSO₄. The optimum parameters for the liquid fermentation were as follows: temperature, 25 °C; cultivation time, 6 d; initial pH, 8.0; volume of medium, 150 mL; and rotary speed, 180 rpm. GREP content and dry cell weight in optimised conditions were 540.1 ± 15.9 mg/L and 8.2 ± 0.3 g/L, respectively. GREP content under the optimised conditions was 2.5 times than that under the basic culture medium and initial conditions. GREP demonstrated positive antioxidant potential on superoxide anion radical, 1,1-diphenyl-2-picrylhydrazyl, and hydroxyl radical scavenging, and reducing power.     

Reaction center complex from an aerobic photosynthetic bacterium,Erythrobacter species OCh 114

A photosynthetic reaction center complex has been purified from an aerobic photosynthetic bacterium, Erythrobacter species OCh 114. The reaction center was solubilized with 0.45% lauryldimethylamine N-oxide and purified by DEAE-Sephacel column chromatography. Absorption spectra of both reduced and oxidized forms of the reaction center were very similar to those of the reaction center from Rhodopseudomonas sphaeroides R-26 except for the contributions due to cytochrome and carotenoid. 1 mol reaction center contained 4 mol bacteriochlorophyll a, 2 mol bacteriopheophytin a, 4 mol cytochrome c-554, 2 mol ubiquinone-10, and carotenoid. The reaction center consisted of four different polypeptides of 26, 30, 32 and 42 kDa. The last one retained heme c. Absorbance at 450 nm oscillated with the period of two on consecutive flashes. The light-minus-dark difference spectrum had two peaks at 450 nm and 420 nm, indicating that odd flashes generated a stable ubisemiquinone anion and even flashes generated quinol. o-Phenanthroline accelerated the re-reduction of flash-oxidized reaction centers, indicating that o-phenanthroline inhibited the electron transfer between Q_A and Q_B. The cytochrome (cytochrome c-554) in the reaction center was oxidized on flash activation. The midpoint potential of the primary electron acceptor (Q_A) was determined by measuring the extent of oxidation of cytochrome c-554 at various ambient potentials. The mid-point potential of Q_A was −44 mV, irrespective of pH between 5.5 and 5.9.