Effects of divalent metal cations on lovastatin biosynthesis from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> in chemically defined medium

Effects of six divalent metal cations: Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺, Cu²⁺and Mn²⁺ on fungal cell growth and lovastatin biosynthesis were investigated by submerged cultivation of Aspergillus terreus in a modified chemically defined medium. The influences of different initial concentrations of the above six metal cations were also examined at 1, 2, and 5 mM, respectively. Cu²⁺ apparently inhibited the cell growth, but had no influence on biosynthesis of lovastatin. All of Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺ and Mn²⁺ promoted the cell growth and lovastatin biosynthesis in different extents. The highest biomass of 13.8 ± 0.5 g l⁻¹ and specific lovastatin titres of 49.2 ± 1.4 mg gDCW⁻¹ were obtained at the level of 2 and 5 mM in the presence of Zn²⁺, respectively. The values were improved double and 14.4-fold. Excess Zn²⁺ inhibited the cell growth, but enhanced lovastatin biosynthesis with an increment of 17.6 mg l⁻¹ per mM. The interactions of all metal cations slightly inhibited the lovastatin production comparing with the existence of Zn²⁺, Fe²⁺ and Mg²⁺ solely, yet remarkably improved the cell growth. These results suggest that the divalent metal ions Zn²⁺ or Fe²⁺ influence the production by regulating the action of key enzymes such as LovD or LovF in lovastatin biosynthesis.     

Levels and sub-cellular distribution of physiologically important metal ions in neuronal cells cultured from chick embryo cerebral cortex

Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺, and Cu content of neurons from chick embryo cortex cultivated in chemically defined serum free growth medium was determined by energy dispersive X-ray fluorescence and atomic absorption spectroscopy. The intracellular volume of cultured neurons was determined to be 2.73 l/mg. Intracellular Mn²⁺, Fe²⁺, Zn²⁺, and Cu²⁺ in the cultivated neurons were 100–200 times the concentrations in the growth medium: Mg²⁺ and Ca²⁺ were 0.9 and 1.7 mM respectively, around 20 fold higher than in growth medium. Mg²⁺, Fe²⁺, Cu²⁺ and Zn²⁺ concentrations in neurons were in the range of ca. 300–600 M, approximately 2–3 times the values previously reported in glial cells; Ca²⁺ and Mn²⁺ content of the neurons were higher by 5 and 10 fold respectively compared to glial cells. In neurons, the subcellular distribution of Fe²⁺, Cu²⁺, and Mn²⁺ follows the rank order: cytosol>microsomes>mitochondria; for Zn²⁺ the distribution differs as following: cytosol >mitochondria>microsomes. Determination of the superoxide dismutase activities in the cultivated neurons indicated that the Mn²⁺ linked activity predominates whereas, the Cu-Zn dependent enzyme is dominant in glial cells. Enrichment of the culture medium with Mn²⁺ to 2.5 M enhanced the Mn-SOD by approximately 33% but Cu²⁺–Zn²⁺ enzyme activity was not modified. The high Mn²⁺ content, the capacity to accumulate Mn²⁺, and the predominancy of the Mn–SOD form observed in neurons is in accord with a fundamental functional role for this metal ion in this type of brain cells.     

Extracellular Ribonuclease Production by Rhizopus stolonifer: Influence of Metal Ions

A strain of Rhizopus stolonifer produced high levels of extracellular ribonuclease (RNase) when grown on YPG (yeast extract, peptone, glucose) medium. Influence of various medium components on the production of extracellular RNase activity showed that divalent metal ions had a marked effect on growth and enzyme production. Maximum enzyme activity (3000 U/ml) was obtained in 5 days when the culture was grown in YPG medium containing Mg²⁺ (12 mM), Mn²⁺, and Fe²⁺ (2 ppm each). Inorganic phosphate, however, repressed enzyme production. Antibodies raised against the purified extracellular RNase were then used to establish the relationship between intra- and extracellular enzymes.     

Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH₄Cl, urea, and ammonium citrate); and metal ions (Zn²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ca²⁺, and K⁺). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg²⁺ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg²⁺. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Effect of nutrients on the accumulation of glutamyl endopeptidase in the culture liquid ofBacillus intermedius 3–19

The effect of nutrients and growth conditions on the accumulation of glutamyl endopeptidase in the culture liquid ofBacillus intermedius 3–19 was studied. Glucose and other readily metabolizable carbon sources were found to suppress the production of the enzyme, whereas inorganic phosphate and ammonium cations enhanced it. Protein substrates, such as casein, gelatin, and hemoglobin, did not affect enzyme production. Some bivalent cations (Ca²⁺, Mg²⁺, Co²⁺) increased the production of glutamyl endopeptidase, but others (Zn²⁺, Fe²⁺, Cu²⁺) acted in the opposite way. The rate of enzyme accumulation in the culture liquid increased as the growth rate of the bacterium decreased, so that the maximum enzyme activity was observed in the stationary growth phase. Based on the results of this investigation, an optimal medium for the maximum production of glutamyl endopeptidase byB. intermedius 3–19 was elaborated.     

Inhibitory effect of ethylenediaminetetraacetate (EDTA) on the porcine lymphocyte response to mitogens and reversal of the effect with metal ions

The effect of ethylenediaminetetraacetate (EDTA) on the mitogen response of porcine lymphocytes and the role of metal ions in reversal of the inhibitory effect of EDTA were determined. Porcine lymphocyte responses to mitogens were totally suppressed when serum used to supplement Ca²⁺, Mg²⁺-free minimum essential medium (MEM) was dialyzed against saline or saline with 0.2 or 0.60 mM EDTA, but the responses were only partially reduced when the same serum was added to RPMI-1640 medium. The inhibition observed in MEM could be reversed by adding 1×10⁻³ M Ca²⁺ and 1×10⁻³ M Mg²⁺ to the dialyzed serum. Serum treated directly with 0.60 mM EDTA completely suppressed blastogenesis in lymphocyte cultures maintained in RPMI-1640 or Ca²⁺, Mg²⁺ free MEM. The inhibitory effect of EDTA-treated serum could be completely reversed by adding Zn²⁺ or a combination of Zn²⁺ with other cationic ions, or partially reversed by adding Ni²⁺ or Fe³⁺. Zn²⁺ was the most effective ion, in that it was the only ion that, when alone added to the serum, could completely restore lymphocyte responses to phytohemagglutinin (PHA) or pokeweed mitogen (PWM).     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。     

Altering the Divalent Metal Ion Preference of RNase E

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg²⁺ and Mn²⁺ will support significant rates of activity in vitro against natural RNAs, with Mn²⁺ being preferred. Both Mg²⁺ and Mn²⁺ also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni²⁺ and Zn²⁺ permitted low levels of activity, while Ca²⁺, Co³⁺, Cu²⁺, and Fe²⁺ did not. A mutation to one of the residues known to chelate Mg²⁺, D346C, led to almost complete loss of activity dependent on Mg²⁺; however, the activity of the mutant enzyme was fully restored by the presence of Mn²⁺ with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO₄, implying that RNase E relies on Mg²⁺ exclusively in vivo.     

Spatial distributions and net deposition rates of mineral elements in the elongating wheat (Triticum aestivum L.) leaf under saline soil conditions

Wheat leaf growth is known to be spatially affected by salinity. The altered spatial distribution of leaf growth under saline conditions may be associated with spatial changes in tissue mineral elements. The objective of this study was to evaluate the spatial distributions of mineral elements and their net deposition rates in the elongating and mature zones of leaf 4 of the main stem of spring wheat (Triticum aestivum L. cv. Lona) during its linear growth phase under saline soil conditions. Plants were grown in an illitic-chloritic silty loam with 0 and 120 mM NaCl. Three days after emergence of leaf 4, sampling was begun at 3 and 13 h into the 16-h light period. Spatial distributions of fresh weight (FW), dry weight (DW), and Na⁺, K⁺, Cl⁻, NO⁻ ₃, Ca²⁺, Mg²⁺, total P, and total N in the elongating and mature tissues were determined on a millimeter scale. The patterns of spatial distribution of Na⁺, Cl⁻, K⁺, NO₃ ⁻, and Ca²⁺ in the growing leaves were affected by salinity, while those of Mg²⁺, total P, and total N were not. Sodium, K⁺, Cl⁻, Ca²⁺, Mg²⁺, and total N concentrations (mmol · kg⁻¹ FW) were consistently higher at 120 mM NaCl than at 0 mM NaCl along the leaf axis from the leaf base, whereas NO₃ ⁻ concentration was lower at 120 mM NaCl. Deposition rates of all nutrients were greatest in the elongation zone. The elongation zone was the strongest sink for mineral elements in the leaf tissues. Local net deposition rates of Na⁺, Cl⁻, Ca²⁺, and Mg²⁺ (mmol · kg⁻¹ FW · h⁻¹) in the most actively elongating zone were enhanced by 120 mM NaCl, whereas for NO₃ ⁻ this was depressed. The lower supply of NO⁻ ₃ to growing leaves may be responsible for the inhibition of growth under saline conditions. Higher tissue concentrations of Na⁺ and Cl⁻ may cause ion imbalance but probably did not result in ion toxicity in the growing leaves. Potassium, Ca²⁺, Mg²⁺, total P, and total N are less plausibly responsible for the reduction in leaf growth in this study. Higher tissue K⁺ and Ca²⁺ concentrations at 120 mM NaCl are probably due to the presence of high Ca²⁺ in the soil of this study. Received: 13 March 1997 / Accepted: 9 June 1997     

Influence of some metal ions on the lipid content and arachidonic acid production byMortierella sp.

The influence of Ca²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ ions on lipid accumulation, fatty acid composition and arachidonic acid (ARA) production byMortierella sp. S-17 was investigated. A beneficial effect of Mn²⁺ in the concentration range of 2–500 mg/L on lipogenesis was observed. The other elements at about 1 g/L repressed lipid accumulation and ARA yield. The highest yield of ARA (723 mg per liter or 148 mg per gram of dry mycelium) after incubation of the fungus in a glucose medium in the presence of 2 mg Mn²⁺ per liter was obtained. A strong inhibitory effect of Fe²⁺ (above 40 mg/L) on ARA formation was observed.     

Ion antagonism in phytochrome-mediated calcium-dependent germination of turions of Spirodela polyrhiza (L.) Schleiden

The light-dependent germination response of turions (resting fronds) is mediated by phytochrome and requires the presence of Ca²⁺ in the medium (K.-J. Appenroth and H. Augsten, 1990, Photochem. Photobiol. 52: 61–65). The Ca²⁺ requirement of germination is apparent only in the presence of exogenous Mg²⁺. A competitive ion antagonism was demonstrated between Ca²⁺ and Mg²⁺ in this physiological response; Mg²⁺ could also be replaced by Ba²⁺ or Sr²⁺. Without exog-enous Mg²⁺, a Ca²⁺ concentration as low as 0.9 μM fulfilled the Ca²⁺ requirement. This type of ion antagonism resembled the competitive Ca/Mg interaction reported previously for calcium-binding proteins. The physiological response was blocked by inhibitors of Ca²⁺ uptake (verapamil, La³⁺). It was concluded that uptake of Ca²⁺ from the external medium is an essential step in the phytochrome-mediated germination of turions. The results are in agreement with the assumption that the uptake of Ca²⁺ is blocked at the side of entry by other alkaline earth ions. Treatment of turions with Mg²⁺ (1 mM) for 24 h at varying times after the red light pulse in otherwise virtually Ca²⁺-free KNO₃ solution resulted in a response similar to a Ca²⁺ step-down treatment. This is in agreement with the assumption that the Ca²⁺- and the Mg²⁺-sensitive periods coincide. The ion interaction described here represents the first photophysiological example in plants of an antagonistic effect between Ca²⁺ and Mg²⁺ similar to that which occurs in vitro with calmodulin. Received: 12 June 1998 / Accepted: 28 December 1998     

Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+

Effects of Cu²⁺ on a non-specific conductance and H⁺-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu²⁺-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q₁₀ for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H₂O₂, 10 mmol/l ascorbate, 100 μg/ml superoxide dismutase, and 100 μg/ml catalase) did not alter Cu²⁺-induced changes in the plasma membrane conductance, suggesting that the effect of Cu²⁺ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca²⁺-channel antagonists (nifedipine, Zn²⁺, Cd²⁺, Fe²⁺, Ni²⁺) inhibited the Cu²⁺-induced non-specific conductance increase. This suggests that changes in Ca²⁺ influx underlie this effect of Cu²⁺. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu²⁺-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H⁺-ATPase activity with half-maximal inhibition occurring at about 5–20 μmol/l and full inhibition at about 100–300 μmol/l. The Hill coefficient of Cu²⁺ inhibition of the H⁺-ATPase was close to two. Received: 8 December 1999 / Accepted: 16 August 2000     

Influence of cobalt and other microelements on the production of betalains and the growth of suspension cultures of Beta vulgaris

The effects of six microelements (Cu²⁺, Mn²⁺, Fe²⁺, Mo²⁺, Zn²⁺, Co²⁺) on the production of betalains and the growth of suspension cultures of Beta vulgaris were studied. The increase of Co²⁺ from 1–5 M resulted in a 60% increment on the production of betalains. A positive effect of this divalent ion was only accomplished when it was added at the beginning of the culture. This was related to a doubling in the specific betalains production rate compared to B5 control medium. No effects on cell growth and ratio of betacyanines to betaxanthines were observed. Mo²⁺, Fe²⁺ and Cu²⁺ presented a positive but less marked effect, while the increase of Mn²⁺ did not show effects on the production of betalains compared to B5 control medium.     

Effects of Metals on 3-Acetyldeoxynivalenol Production by Fusarium graminearum R2118 in Submerged Cultures

The effects of selected metals (Mg²⁺, Mn²⁺, Zn²⁺, and Fe²⁺) on 3-acetyldeoxynivalenol (3-ADN) production by Fusarium graminearum R2118 and on its mycelial growth were investigated by using a two-stage submerged-culture technique. In certain concentrations ranges, Mg²⁺ and Fe²⁺ stimulated growth but suppressed 3-ADN production; at other concentrations, Mg²⁺, Fe²⁺, and Zn²⁺ suppressed growth but stimulated 3-ADN production. In contrast, Mn²⁺ stimulated growth but totally inhibited 3-ADN production at all concentrations tested. In general, the production of 3-ADN was inversely related to the growth rate of the fungus with these metals. Mn²⁺ appears to be a crucial factor regulating the onset of 3-ADN biosynthesis.     

Nuclease Stn α from <Emphasis Type="Italic">Streptomyces thermonitrificans</Emphasis>: Characterization of the Associated Adenylic Acid Preferential Ribonuclease Activity

Nuclease Stn α from Streptomyces thermonitrificans hydrolyses DNA and RNA at the rate of approximately 10:l. The optimum pH and temperature for RNA hydrolysis were 7.0 and 45°C. The RNase activity of nuclease Stn α had neither an obligate requirement of metal ions nor was it activated in the presence of metal ions. The enzyme was inhibited by Zn²⁺, Mg²⁺, Co²⁺, and Ca²⁺; inorganic phosphate; pyrophosphate; NaCl; KCl; and metal chelators. It was stable at high concentrations of urea but susceptible to low concentrations of Sodium dodecyl sulfate and guanidine hydrochloride. The rates by which nuclease Stn α hydrolysed polyribonucleotides occurs in the order of poly A >> RNA >> poly U > poly G > poly C. The enzyme cleaved RNA to 3′ mononucleotides with preferential liberation of 3′AMP, indicating it to be an adenylic acid preferential endonuclease.     

Production of pyruvic acid from 1,2-propanediol byPseudomonas sp. strain TB-135 which does not require thiamine

Summary Pseudomonas sp. strain TB-135 produces D-lactic acid from 1,2-propanediol (1,2-PD) and requires Ca²⁺, Mg²⁺ and Fe²⁺ for growth but does not require thiamine. The strain produced pyruvic acid only under Fe²⁺-deficient conditions and the addition of Cu²⁺ increased pyruvic acid production. Under optimal conditions (0.03 ppm of FeSO₄ and 0.5ppm of CuSO₄), the strain accumulated 14 mg pyruvic acid par ml after 3 days of cultivation. The thiamine concentration in the cells grown on Fe²⁺-deficient medium was about 6% of that in the cells grown on Fe²⁺-sufficient medium, though pyruvate dehydrogenase (EC 1.2.4.1) activities of both types of cells were the same. We conclude that the low thiamine content of the cells is responsible for the acid production.     

Purification and Characterization of Thermophilic Xylanase Isolated from the Xerophytic-<Emphasis Type="Italic">Cereus pterogonus</Emphasis> sp.

A thermo stable xylanase was purified and characterized from the cladodes of Cereus pterogonus plant species. The enzyme was purified to homogeneity by ammonium sulfate (80%) fractionation, ion exchange and size exclusion chromatography. The enzyme showed a final specific activity of 216.2 U/mg and the molecular mass of the protein was 80 KDa. The optimum pH and temperature for xylanase activity were 5.0 and 80 °C, respectively,. With oat spelt xylan as a substrate the enzyme yielded a Km value of 2.24 mg/mL and a Vmax of 5.8 μmol min⁻¹ mg⁻¹. In the presence of metal ions (1 mM) such as Co²⁺,Mn²⁺, Ni²⁺, Ca²⁺ and Fe³⁺ the activity of the enzyme increased, where as strong inhibition of the enzyme activity was observed with the use of Hg²⁺, Cd²⁺, Cu²⁺, while partial inhibition was noted with Zn²⁺ and Mg²⁺. The substrate specificity of the xylanase yielded maximum activity with oat spelt xylan.     

Novel synthesis of mannosamine conjugated magnetic nanoparticles for purification and stabilization of human lysosomal β-mannosidase

Human β-mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic (Fe₃O₄) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. Fe₃O₄ nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with D-mannosamine by cross linker glutaraldehyde and confirmed by FTIR spectroscopy. In FTIR analysis, the peaks appeared at 2,356.6 cm⁻¹ for −N = CH linkage and at 3,378.4 cm⁻¹, 3,664.9 cm⁻¹ for −OH groups confirmed the conjugation of D-mannosamine with Fe₃O₄ nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the K_m and Vmax values were 2.51 mM and 0.315 μM/min/mg, respectively. The purification fold was 329 with 68% yield. The optimal activity was at pH 5.0 and 75% activity was stable in 20% glycerol at 4°C. The enzyme activity was inhibited by Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Mo²⁺, Ag⁺¹, iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by Pb²⁺, Co²⁺, EDTA, DTT, and β-mercaptoethanol. The activity was not affected by Mg²⁺, Mn²⁺, Sn²⁺, Ca²⁺, Fe³⁺, PMSF, Triton X-100, D-mannosamine, D-mannose, D-mannitol, D-glucose, and D-fructose. The homogeneity of MANB enzyme was further confirmed by 2D-PAGE and immunoblot. This is the first novel report of conjugation of D-mannosamine with Fe₃O₄ nanoparticles for purification of human MANB enzyme.     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.