Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58-60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C alpha-distance 1.2 A). However, this peroxidase includes a Mn2+ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn2+. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.     

Characterization of a novel manganese peroxidase-lignin peroxidase hybrid isozyme produced by Bjerkandera species strain BOS55 in the absence of manganese

A novel manganese-dependent peroxidase (MnP) isozyme produced in manganese-free cultures of Bjerkandera sp. strain BOS55 was purified and characterized. The production of the enzyme was greatly stimulated by the exogenous addition of various physiological organic acids such as glycolate, glyoxylate, and oxalate. The physical properties of the enzyme are similar to those of MnP isozymes from different white rot fungi (Mr = 43,000, pI 3.88, and epsilon407 nm = 123 mM-1 cm-1). The Bjerkandera MnP was efficient in the oxidation of Mn(II), as indicated by the kinetic constants (low Km of 51 microM and turnover number of 59 s-1). Furthermore, the isozyme was able to oxidize various substrates in the absence of manganese, such as 2,6-dimethoxyphenol, guaiacol, ABTS, 3-hydroxyanthranilic acid, and o- and p-anisidine. An interesting characteristic of the isozyme was its ability to oxidize nonphenolic substrates, veratryl alcohol and 1,4-dimethoxybenzene, without manganese addition. The affinity for veratryl alcohol (Km = 116 microM) and its turnover number (2.8 s-1) are comparable to those of lignin peroxidase (LiP) isozymes from other white rot fungi. Manganese at concentrations greater than 0.1 mM severely inhibited the oxidation of veratryl alcohol. The results suggest that this single isozyme is a hybrid between MnP and LiP found in other white rot fungi. The N-terminal amino acid sequence showed a very high homology to those of both MnP and LiP isozymes from Trametes versicolor.     

Control of buccal peroxidases by a bacterial NADH-hypothiocyanite oxidoreductase

Oral peroxidases (myeloperoxidase, sialoperoxidase) catalyze thiocyanate peroxidation into hypothiocyanite which is bacteriostatic or bactericidal against numerous bacterial species. NADH-hypothiocyanite-oxidoreductase is thought to protect bacteria which can express it; up to now, this enzyme activity was never purified. The present study analyzes, on one hand, the susceptibility of periodontal bacteria against hypothiocyanite and, on the other hand, proposes a purification design for the NADH-hypothiocyanite-oxidoreductase from Streptococcus sanguis, a commensal micro-organism of dental surfaces. The data suggest the importance of the bacterial biofilm on dental surfaces for production of antiseptic oxidants and for the control of their toxicity.     

湖南省工业废水中铁锰排放标准研究

通过对湖南省典型铁锰污染区域的钢铁、有色金属、电解锰及化工企业生产工艺、废水处理技术与污染物排放现状及受污染河流环境质量现状进行调查的基础上,参照国外废水中铁锰污染物排放限值,规定湖南省工业废水中现有企业总铁的排放标准限值为10 mg/L,新建企业废水中总铁排放标准限值为5 mg/L;湖南省工业废水中总锰的排放标准限值为1 mg/L.     

Purification and cloning of a thermostable manganese catalase from a thermophilic bacterium

We have purified a heat-stable catalase from a thermophilic bacterium, Thermus species strain YS 8-13. The enzyme was purified 160-fold from crude cellular extracts and possessed a specific activity of 8000 units/mg at 65 degrees C. The purified enzyme displayed the highest activity at pH 7 to 10 and temperatures around 85 degrees C. The catalase was determined to be a manganese catalase, based on results from atomic absorption spectra and inhibition experiments using sodium azide. The enzyme was composed of six identical subunits of molecular weight 36,000. Amino acid sequences determined from the purified protein were used to design oligonucleotide primers, which were in turn used to clone the coding gene. The nucleotide sequence of a 1.4-kb fragment of Thermus sp. YS 8-13 genomic DNA containing a 909-bp open reading frame was determined. The gene encoded a 302-residue polypeptide of deduced molecular weight 33,303. The deduced amino acid sequence displayed a region-specific homology with the sequences of the manganese catalase from a mesophilic organism, Lactobacillus plantarum.     

Cobalt extraction and concentration from manganese wad by leaching and precipitation

Manganese wad often contains significant amounts of nickel, cobalt and copper, and it may represent a future source of these metals. The mineralogy and extractive metallurgy of a number of Australian wads are being examined with regard to producing a high-grade non-ferrous metal sulphide suitable for further processing. This paper describes the general approach taken, and some specific results obtained when a cobalt-rich wad from Queensland was leached with sulphur dioxide. The rate of dissolution is affected by the feed composition, particle size and sulphur dioxide flowrate. Under the conditions used, > 90% Co, Cu and Ni extraction is achieved in about 8 h. The Co, Cu and Ni can be readily precipitated from the clarified leachate by injection of hydrogen sulphide. The product contains about 60% (Co + Cu + Ni) with less than 0.1% Mn. As leaching is non-selective with respect to manganese, it will probably be necessary to recover and recycle the sulphur dioxide associated with the dissolved manganese when the ground wad is processed on a commercial scale.     

Purification and characterization of cysteine protease from Pleurotus ostreatus

Cysteine protease activity in mycelial culture increased 7.7-fold after fruit body formation in Pleurotus ostreatus, using the Leu pNA (LPNA) cleavage assay. The enzyme was purified from fruit bodies and its M(r) was 97,000 by gel filtration and 48,500 by SDS-PAGE, indicating that it is a dimer. The enzyme was sensitive to iodoacetic acid, p-chloromercuribenzoate, N-ethylmaleimide, and HgCl2. The sequence of the first 9 N-terminal amino acids of cysteine protease was ASGLXXAIL.     

Characterization of laccases and peroxidases from wood-rotting fungi (family Coprinaceae)

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60 degrees C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.     

Single cell extraction of zinc and manganese dioxide from zinc sulphide concentrate and manganese ores

The hydrometallurgical processing of zinc sulphide concentrates with sulphuric acid in the presence of manganese dioxide (manganese ore has been employed) and subsequent electrolytic co-deposition of cathodic zinc metal and anodic manganese dioxide is described.The influence of various parameters on the reaction

$\text{ZnS + MnO}{}_2\text{+ 2H}{}_2\text{SO}{}_4\text{= ZnSO}{}_4\text{+ MnSO}{}_4\text{+ S}{}^0\text{+ 2H}{}_2\text{O}$

has been studied. Optimum conditions for rapid and efficient reaction have been determined.The simultaneous electrowinning of zinc at the cathode and γ-MnO₂ at the anode from the leach liquor was studied. The effects of variation of current density, temperature, electrolyte composition etc. have been described in detail. During leaching 99% extraction of zinc, 98% of manganese, and 96% liberation of elemental sulphur was achieved. 80–90% anodic and cathodic current efficiencies can be obtained under optimum conditions with impurity levels of only a trace of manganese in the zinc deposit and vice-versa.The anodically deposited manganese dioxide was the γ-battery active variety and was found to be satisfactory.The results indicate the potential for the development of a technique for zinc and manganese dioxide production in a single cell.     

印尼某红土矿硫酸加压浸出矿物组成变化研究

印尼苏拉威西岛La-paopao矿区红土镍矿储量约7326万吨,含镍约为1.25％,以此红土镍矿为对象,系统分析了矿样中主要矿物的种类、赋存状态及产出特征.结果 表明:矿样含Fe (40.15％),Ni(1.42％),Co(0.15％),Mg(0.37％),SiO2(6.92％)(质量分数),是典型的褐铁型红土镍矿;组...     

转炉炼钢加锰矿提高终点锰含量的试验研究

介绍了锰矿还原的机理和转炉吹炼过程中加入锰矿的试验情况和效果,对影响终点残锰含量的因素进行了分析研究,提出了在转炉吹炼过程中加入锰矿来提高终点残锰的技术措施。     

Kinetics of manganese oxide reduction from basic slags by carbon dissolved in liquid iron

The reduction of manganese oxide from a basic slag by carbon dissolved in liquid iron is a slow reaction, failing to approach equilibrium closely in 20 hr. Furthermore, the rate of stirring has no apparent effect on the reaction rate. This identifies the rate-controlling step as a chemical reaction at the interface. Only the model for the reactionO ²⁻ =O + 2e ⁻ gave a consistent interpretation as the melt geometry, and concentration of manganese oxide and carbon were varied. The rate constant for this reaction was found to be 1.28 × 10⁻⁵ mole per sq cm per min at 155O°C. The effect of temperature is substantial with a calculated energy of activation for the system of 25 kcal per mole. Formerly Graduate Student, The University of Michigan This paper is based on a portion of a thesis submitted by W. L. DAINES in partial fulfillment of the requirements for the degree Doctor of Philosophy at The University of Michigan.     

Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism

Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase [CCP(MI)] and horseradish peroxidase (HRP) in their ferric forms. CCP(MI) unfolds in urea and in guanidine hydrochloride (GdHCl) at pH 7.0, while HRP loses its secondary structure only in the presence of GdHCl. CCP(MI) unfolds in urea by two distinct steps as monitored by fluorescence, but the loss of its secondary structure as monitored by UV/CD occurs in a single step between 3.4 and 5 M urea and 1.5 and 2.5 M GdHCl. The localized changes detected by fluorescence involve the CCP(MI) heme cavity since the Soret maximum red-shifts from 408 to 416 nm, and the heme CD changes examined in urea are biphasic. The polypeptide of HRP also loses secondary structure in a single step between 1.2 and 2.7 M GdHCl as monitored by UV/CD, and a fluorescence-monitored transition involving conformational change in the Trp117-containing loop occurs above 4 M GdHCl. Free energies of denaturation extrapolated to 0 M denaturant (delta Gd,aq) of approximately 6 and approximately 4 kcal/mol were calculated for CCP(MI) and HRP, respectively, from the UV/CD data. The refolding mechanisms of the two peroxidases differ since heme capture in CCP(MI) is synchronous with refolding while apoHRP captures heme after refolding. Thus, the denatured form of apoHRP does not recognize heme and has to correctly refold prior to heme capture. The half-life for unfolding of native HRP in 6 M GdHCl is slow (519 s) compared to that for CCP(MI) (14.3 s), indicating that HRP is kinetically much more stable than CCP(MI). Treatment with EDTA and DTT greatly destabilizes HRP, and unfolding in 4 M GdHCl occurs with t1/2 = 0.42 s.     

Production of manganese magnesia sinter using enriched nikopol manganese ore and magnesia-silicate slag from ferronickel production

The industrial introduction of a new production technology for manganese magnesia sinter (7–10% MgO) at PAO NZF is considered. In this technology, the raw materials are enriched Nikopol manganese ore and magnesia-silicate slag (28–30% MgO, 50–52% SiO₂) from the production of ferronickel (15–20% Ni) at Pobuzhsk Ferronickel Works on the basis of imported nickel ore with magnesia-bearing barren rock. Data are presented regarding the microstructure of the sinter, the chemical composition of the phase-mineral aggregations in the sinter, and the mechanical properties of standard and experimental sinter samples.     

Purification of peroxidases and separation of isoperoxidases from various plant species (author's transl)

The partial purification of peroxidase (EC.1.11.1.7) and separation of isoperoxidases by disc electrophoresis from Cucurbita Pepo L., Phaseolus vulgaris L., Cicer arietinum L. and Hordeum, Secale and Triticum sp., have been studied. Peroxidase from fruit of pumpkin and from 6-day-old coleoptiles of French bean and chick pea has been partially purified, 128-, 174-, and 140-fold, respectively. The apparent Km at the optimum pH were: pumpkin (epicarp.), 2.7 X 10(-4) M; barley, common rye and wheat (primary leaves, in all cases), 1.4 X 10(-5), 1.2 X 10(-5) and 3.1 X 10(-5) M, respectively. Isoperoxidases have been separated by disc electrophoresis on 7% polyacrylamide gel and stained with p-phenylenediamine. Differences in patterns of anodic and cathodic isozymes were observed: 3 isozymes from fruits of pumpkin, 4 from French bean, 4 from chick pea, 11 from leaves of barley, 10 from leaves of common rye and 9 from leaves of wheat.