Purification and characteristics of a low-molecular-weight peptide possessing oxidative capacity for phenol from Phanerochaete chrysosporium

A new low-molecular-weight peptide with phenol oxidase activity, named Pc factor, was isolated and purified from liquid culture of a white-rot basidiomycete Phanerochaete chrysosporium. Its molecular weight was about 600 Da estimated by gel-filtration. Three amino acids Glu, Gly and Val were detected in hydrolysate. Absorption peaks corresponding to amino acids and peptide were observed by UV and IR spectra analysis. And the signal of Cα of amino acid was also detected by ¹³C-NMR method. Pc factor had high thermostability and remained active in weakly alkalescent pH range. It could chelate Fe³⁺ and reduce it to Fe²⁺, but no hydroxyl radical HO▪ could be detected during the reaction process. It could oxidize phenolic lignin-model compounds such as 2,6-dimethoxyphenol (2,6-DMP), 2,2¢-azinobis (3-ethylbenzathiazoline-6-sulfinic acid) (ABTS) and syringaldazine in the absence of Mn²⁺ and H₂O₂. These characteristics differed greatly from those of manganese peroxi-dases. The oxidative catalysis of Pc factor can be enhanced by certain metal ions such as Cu²⁺ and Mn²⁺ etc., and O2 molecule was necessary for this reaction. In summary, Pc factor may function as an electron carrier in this novel oxidation-reduction system.     

A peptide-mediated and hydroxyl radical HO*-involved oxidative degradation of cellulose by brown-rot fungi

A special low-molecular-weight peptide named Gt factor, was isolated and purified from the extracellular culture of brown-rot fungi Gloeophyllum trabeum via gel filtration chromatography and HPLC. It has been shown to reduce Fe³⁺ to Fe²⁺. Electron paramagnetic resonance (EPR) spectroscopy revealed Gt factor was able to drive H₂O₂ generation via a superoxide anion O₂ ^.- intermediate and mediate the formation of hydroxyl radical HO^. in the presence of O₂. All the results indicated that Gt factor could oxidize the cellulose, disrupt the inter- and intrahydrogen bonds in cellulose chains by a HO^. -involved mechanism. This resulted in depolymerization of the cellulose, which made it accessible for further enzymatic hydrolysis.     

In vitro anti- and pro-oxidative effects of natural polyphenols

The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems utilizing ethyl linoleate and 2′-deoxyguanosine (2′-dG) as oxidative substrates, and a Fenton reaction (H₂O₂, Fe²⁺) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2′-dG assay, oxidation was closely related to H₂O₂ generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn²⁺ and Cu²⁺, but not by Zn²⁺ or Co²⁺. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe³⁺ to the active Fe²⁺ state.     

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.     

Flux of reduced chemical constituents (Fe2+, Mn2+, NH inf4 sup+ and CH4) and sediment oxygen demand in Lake Erie

Sediment pore water concentrations of Fe²⁺, Mn²⁺, NH _inf4 ^sup+ and CH₄ were analyzed from both diver-collected cores and anin situ equilibration device (peeper) in Lake Erie's central basin. Sediment oxygen demand (SOD) was measured at the same station with a hemispheric chamber (including DO probe and recorder) subtending a known area of sediments. The average SOD was 9.4 mM m⁻² day⁻¹ (0.3 g m⁻² day⁻¹). From pore water gradients within the near-surface zone, the chemical flux across the interface was calculated indirectly using Fick's first law modified for sediments. These calculations, using core and peeper gradients, always showed sediment loss to overlying waters, and variations between the two techniques differed by less than an order of magnitude for Fe²⁺ and CH₄. The transport of these reduced constituents can represent a sizeable oxygen demand, ranging from less than 1% for Fe²⁺ and Mn²⁺ to as high as 26% for NH _inf4 ^sup+ , and 30% for CH₄. The average flux of these constituents could account for about a third of the SOD at the sediment-water interface of this station.     

Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerR_BS protein, represses target genes when bound to either Mn²⁺ or Fe²⁺ as corepressor, but only the Fe²⁺-bound form responds to H₂O₂. The orthologous protein in the human pathogen Staphylococcus aureus, PerR_SA, plays important roles in H₂O₂ resistance and virulence. However, PerR_SA is reported to only respond to Mn²⁺ as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H₂O₂ sensing. Here we demonstrate that PerR_SA uses either Fe²⁺ or Mn²⁺ as corepressor, and that, like PerR_BS, the Fe²⁺-bound form of PerR_SA senses physiological levels of H₂O₂ by iron-mediated histidine oxidation. Moreover, we show that PerR_SA is poised to sense very low levels of endogenous H₂O₂, which normally cannot be sensed by B. subtilis PerR_BS. This hypersensitivity of PerR_SA accounts for the apparent lack of Fe²⁺-dependent repressor activity and consequent Mn²⁺-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerR_SA is directly correlated with virulence, whereas it is inversely correlated with H₂O₂ resistance, suggesting that PerR_SA may be an attractive target for the control of S. aureus pathogenesis.     

Oxidation of manganous pyrophosphate by superoxide radicals and illuminated spinach chloroplasts.

The oxidation of Mn²⁺-pyrophosphate to Mn³⁺ by superoxide (O₂^?) was quantitative as evidenced from the formation of Mn³⁺-pyrophosphate and hydrogen peroxide and from the inhibition by superoxide dismutase. Using the competitive relation between Mn²⁺-pyrophosphate and superoxide dismutase for the O₂^?, the rate constant of Mn²⁺ oxidation was estimated to be about 6 × 10⁶m^?1 s^?1. The oxidation of Mn²⁺-pyrophosphate by illuminated chloroplasts was also indicated to be stoichiometrically induced by O₂^?. In the presence of saturating amounts of the Mn²⁺, a double enhancement of hydrogen peroxide production and triple uptake of oxygen were found, as expected from the oxidation of Mn²⁺-pyrophosphate by O₂^?. Anaerobiosis or superoxide dismutase annuled these increments. We propose that the O₂^? generated as the sole initial step of the Mehler reaction oxidized Mn²⁺-pyrophosphate, and we discuss the role of free manganese in chloroplasts.     

Effect of manganese on ninhydrin color development by amino acids

Addition of microgram quantities (5–10 μg per 2 ml) of Mn²⁺, Fe²⁺, and Mo²⁺ increases the intensity of color developed by amino acids two- to three-fold when reacting with ninhydrin. The lowest limit detected by the increased sensitivity of the reaction is 3 nmol of an amino acid. Cd²⁺, Zn²⁺, and Al³⁺ depress the color formation due to reaction between amino acids and ninhydrin. When chromatograms of amino acids are first sprayed with aqueous manganese chloride and later with ninhydrin or with ninhydrin solution containing manganese, not only is the sensitivity increased, but the stained spots retain their color for longer periods.     

Determination of the growth and solubilization capabilities of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> T1

The growth capability of Trichoderma harzianum Rifaii Tl was tested on Malt Extract and Czapeks Dox agar containing different concentrations of Cu²⁺, Zn²⁺, Mn²⁺, Fe²⁺ and Ca²⁺. The T. harzianum Tl isolate was observed to produce mycelia and spores in various mineral-containing media. It showed the lowest tolerance to Ca²⁺ and the highest tolerance to Fe²⁺. Solubilization capability of T. harzianum Tl for some insoluble minerals via acidification of medium has been tested on MnO₂, CuO, Fe₂O₃ and metallic Zn. T. harzianum Tl was able to solubilize MnO₂ and metallic Zn in a liquid medium.     

Lactobacillus sanfranciscensis CB1: manganese, oxygen, superoxide dismutase and metabolism

The latency phase, growth rate, cell yield and end-products of Lactobacillus sanfranciscensis CB1 were affected by oxygen and the supply of 225 μM Mn²⁺. Mn²⁺ was especially related to the highest substrate consumption. Aerobiosis and Mn²⁺ supply were responsible for the highest superoxide dismutase activity. An auto-inhibitory accumulation of H₂O₂ meant that the survival of air-grown cells supplied with Mn²⁺ rapidly decreased during the stationary phase. As shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Mn²⁺ supply influenced protein expression. As shown by non-denaturating zymograms, Lb. sanfranciscensis CB1 expressed an approximately 12.5-kDa superoxide dismutase, which is probably Mn-dependent. The enzyme was insensitive to H₂O₂ treatment, was not induced by the presence of paraquat under aerobic conditions and was relatively stable at pH 4.0. Sourdoughs that contained high levels of oxygen enhanced cell growth, acidification and acetic acid production by Lb. sanfranciscensis CB1. Received: 24 July 1998 / Received last revision: 11 November 1998 / Accepted: 13 November 1998     

Involvement of ligninolytic enzymes and Fenton-like reaction in humic acid degradation by Trametes sp

Trametes sp. M23, isolated from biosolids compost was found to decompose humic acids (HA). A low N (LN) medium (C/N, 53) provided suitable conditions for HA degradation, whereas in a high N (HN) medium (C/N, 10), HA was not degraded. In the absence of Mn²⁺, HA degradation was similar to that in Mn²⁺-containing medium. In contrast, MnP activity was significantly affected by Mn²⁺. Laccase activity exhibited a negative correlation to HA degradation, while LiP activity was not detected. Thus, ligninolytic enzymes activity could provide only a partial explanation for the HA-degradation mechanism. The decolorization of two dyes, Orange II and Brilliant Blue R250, was also determined. Similar to HA degradation, under LN conditions, decolorization occurred independently of the presence of Mn²⁺. We investigated the possible involvement of a Fenton-like reaction in HA degradation. The addition of DMSO, an OH-radical scavenger, to LN media resulted in a significant decrease in HA bleaching. The rate of extracellular Fe³⁺ reduction was much higher in the LN vs. HN medium. In addition, the rate of reduction was even higher in the presence of HA in the medium. In vitro HA bleaching in non-inoculated media was observed with H₂O₂ amendment to a final concentration of 200 mM (obtained by 50 mM amendments for 4 days) and Fe²⁺ (36 mM). After 4 days of incubation, HA decolorization was similar to the biological treatment. These results support our hypothesis that a Fenton-like reaction is involved in HA degradation by Trametes sp. M23.     

Peroxidation of linoleic acid during the oxidation of phenols by fungal laccase

A system comprising laccase and a suitable phenol such as 4-hydroxybenzoic acid (HBA) or synthetic lignin (DHP) exhaustively peroxidized linoleic acid in acetate buffer. The presence of phenols in lignin was essential since an exhaustively methylated preparation of the same lignin did not support peroxidation. The peroxidation rate was greatly enhanced by Mn²⁺, which was oxidized to Mn³⁺ by laccase/HBA, whereas H₂O₂ inhibited strongly due to rapid reduction of Mn³⁺ by H₂O₂ with concomitant formation of O₂. When acetate was replaced by Mn³⁺–chelating oxalate or malonate, there was no change in peroxidation rates in the absence of Mn²⁺, whereas strong inhibition was observed in the presence of Mn²⁺. In case of malonate part of the inhibition was due to H₂O₂ formation as a result of Mn³⁺ reduction by malonate. These findings suggest that laccase may contribute to fungal lipid peroxidation in vivo thus expanding its role in the biodegradation of lignin and other recalcitrant aromatic compounds.     

Luminescence and energy transfer in Ce3+, Mn2+‐doped K2AEP2O7 (AE = Ca,Sr) phosphor

Pyrophosphates K₂AEP₂O₇ (AE = Ca, Sr) prepared by the classical solid‐state technique and activated with Ce³⁺ are described. Intense emission was observed in K₂AEP₂O₇ (AE = Ca, Sr). The effect of Mn²⁺ co‐doping was studied. The broad emission peak of Mn²⁺ was observed at 534 nm in K₂SrP₂O₇:Ce³⁺ and at 539 nm in K₂CaP₂O₇:Ce³⁺, Mn²⁺. Mn²⁺ emission was greatly enhanced by addition of the sensitizer Ce³⁺ due to efficient energy transfer from Ce³⁺ to Mn²⁺. Copyright © 2015 John Wiley & Sons, Ltd.     

Flash-induced consumption of molecular oxygen on the donor side of photosystem II in Mn-depleted subchloroplast membrane fragments: specific effects of manganese and calcium ions

It has been shown that removal of manganese from the water-oxidizing complex (WOC) of photosystem II (PSII) leads to flash-induced oxygen consumption (FIOC) which is activated by low concentration of Mn²⁺ (Yanykin et al., Biochim Biophys Acta 1797:516–523, 2010). In the present work, we examined the effect of transition and non-transition divalent metal ions on FIOC in Mn-depleted PSII (apo-WOC-PSII) preparations. It was shown that only Mn²⁺ ions are able to activate FIOC while other transition metal ions (Fe²⁺, V²⁺ and Cr²⁺) capable of electron donation to the apo-WOC-PSII suppressed the photoconsumption of O₂. Co²⁺ ions with a high redox potential (E ⁰ for Co²⁺/Co³⁺ is 1.8 V) showed no effect. Non-transition metal ions Ca²⁺ by Mg²⁺ did not stimulate FIOC. However, Ca²⁺ (in contrast to Mg²⁺) showed an additional activation effect in the presence of exogenic Mn²⁺. The Ca²⁺ effect depended on the concentration of both Mn²⁺ and Ca²⁺. The Ca effect was only observed when: (1) the activation of FIOC induced by Mn²⁺ did not reach its maximum, (2) the concentration of Ca²⁺ did not exceed 40 μM; at higher concentrations Ca²⁺ inhibited the Mn²⁺-activated O₂ photoconsumption. Replacement of Ca²⁺ by Mg²⁺ led to a suppression of Mn²⁺-activated O₂ photoconsumption; while, addition of Ca²⁺ resulted in elimination of the Mg²⁺ inhibitory effect and activation of FIOC. Thus, only Mn²⁺ and Ca²⁺ (which are constituents of the WOC) have specific effects of activation of FIOC in apo-WOC-PSII preparations. Possible reactions involving Mn²⁺ and Ca²⁺ which could lead to the activation of FIOC in the apo-WOC-PSII are discussed.     

Requirement of Manganese for Electron Donation of Hydrogen Peroxide in Photosystem II Reaction Center Complex

Mn²⁺ was required for the electron donating reaction from H₂O₂,but not for that from diphenylcarbazide (DPC), in the PS IIreaction center complex which was prepared from spinach chloroplastsby Triton X-100 extraction. The reaction center complex showeda high activity of 2,6-dichloroindophenol (DCIP) photoreductionin the presence of DPC, but a low activity with H₂O₂. The H₂O₂-supportedDCIP photoreduction was suppressed by EDTA and enhanced by asmall amount of Mn²⁺. Ca²⁺ and Mg²⁺ could not replace Mn²⁺.The activation by Mn²⁺ and its binding showed two binding sitesof Mn²⁺ in the reaction center complex, with high (1.5?10⁷ M^–1)and low (1 ? 10⁶ M^–1) binding constants. (Received November 8, 1986; Accepted April 10, 1987)     

Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism

Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO₂, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe²⁺, α-ketoglutarate, O₂ and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO₂, succinate and Fe²⁺, in which Fe²⁺ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO₂ is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO₂ and succinate or after the release of all products, including Fe²⁺, and dehydroascorbate is released before the binding of Fe²⁺. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical.Zn²⁺, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe²⁺, α-ketoglutarate, O₂ and ascorbate respectively, and epinephrine noncompetitively with regard to all cosubstrates. Apparent K_i values are given for the product and other inhibitors.     

De novo synthesis,constitutive expression of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> β-xylanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and partial enzymic characterization

The endo-β-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an α-mating factor in a constitutive expression vector pGAPzαA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast–peptone–dextrose liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed with a yield of 120 units/mL under the flask culture at 28°C for 3 days. The enzyme showed optimal activity at 50°C and pH 2.4–3.4. Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80°C for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60°C for 30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na₂HPO₄-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na⁺, Mg²⁺, Ca²⁺, K⁺, Ba²⁺, Zn²⁺, Fe²⁺, and Mn²⁺) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Amorphous Manganese-Calcium Oxides as a Possible Evolutionary Origin for the CaMn<Subscript>4</Subscript> Cluster in Photosystem II

In this paper a few calcium-manganese oxides and calcium-manganese minerals are studied as catalysts for water oxidation. The natural mineral marokite is also studied as a catalyst for water oxidation for the first time. Marokite is made up of edge-sharing Mn³⁺ in a distorted octahedral environment and eight-coordinate Ca²⁺ centered polyhedral layers. The structure is similar to recent models of the oxygen evolving complex in photosystem II. Thus, the oxygen evolving complex in photosystem II does not have an unusual structure and could be synthesized hydrothermally. Also in this paper, oxygen evolution is studied with marokite (CaMn₂O₄), pyrolusite (MnO₂) and compared with hollandite (Ba_0.2Ca_0.15K_0.3Mn_6.9Al_0.2Si_0.3O₁₆), hausmannite (Mn₃O₄), Mn₂O₃.H₂O, CaMn₃O₆.H₂O, CaMn₄O₈.H₂O, CaMn₂O₄.H₂O and synthetic marokite (CaMn₂O₄). I propose that the origin of the oxygen evolving complex in photosystem II resulted from absorption of calcium and manganese ions that were precipitated together in the archean oceans by protocyanobacteria because of changing pH from ~5 to ~8-10. As reported in this paper, amorphous calcium-manganese oxides with different ratios of manganese and calcium are effective catalysts for water oxidation. The bond types and lengths of the calcium and manganese ions in the calcium-manganese oxides are directly comparable to those in the OEC. This primitive structure of these amorphous calcium-manganese compounds could be changed and modified by environmental groups (amino acids) to form the oxygen evolving complex in photosystem II.     

EPR spectroscopy and catalase activity of manganese-bound DNA-binding protein from nutrient starved cells

DNA-binding proteins from nutrient-starved cells (DPS) protect cells from oxidative stress by removing H₂O₂ and iron. A new class of DPS-like proteins has recently been identified, with DPS-like protein from Sulfolobus solfataricus (SsDPS) being the best characterized to date. SsDPS protects cells from oxidative stress and is upregulated in response to H₂O₂ but also in response to iron depletion. The ferroxidase active site of SsDPS is structurally similar to the active sites of manganese catalase and rat liver arginase. The present work shows that the ferroxidase center in SsDPS binds two Mn²⁺ ions with K _D = (1/K ₁ K ₂)^1/2 = 48(3) μM. The binding constant of the second Mn²⁺ is significantly higher than that of the first, inducing dinuclear Mn(II) cluster formation for all but the lowest concentrations of added Mn²⁺. In competition experiments, equimolar amounts of Fe²⁺ were unable to displace the bound manganese. EPR spectroscopy of the Mn₂ ²⁺ cluster showed signals comparable to those of other characterized dimanganese clusters. The exchange coupling for the cluster was determined, J = −1.4(3) cm⁻¹ (H = −2JS ₁ S ₂), and is within the range expected for a μ_1,1-carboxylato bridge between the manganese ions. Manganese-bound SsDPS showed catalase activity at a rate 10–100 times slower than for manganese catalases. EPR spectra of SsDPS after addition of H₂O₂ showed the appearance of an intermediate in the reaction with H₂O₂.