Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

A peroxidase oxidizing Mn²⁺ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn²⁺-mediated as well as Mn²⁺-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.     

Relative shifts in mobility in anionic peroxidase isoenzymes between stem base and apex of flax genotrophs

Anionic peroxidase isoenzymes, separated on acrylamide gels, were examined in two flax genotrophs and in their reciprocal F₂ hybrids. Isoenzyme 1 exhibited a significant difference in R_m between stem base and apex and there was a gradient of decreasing R_m and activity between base and apex. Isoenzyme 2 displayed only the activity gradient. The parents differed significantly in the R_m's and activities of isoenzymes 1 and 2, and the F₂'s showed complete dominance of the L parent for R_m, with activities being approximately intermediate.     

Changes in peroxidase isoenzymes of Phaseolus mungo hypocotyl cuttings during rooting

A time course study of changes in the pattern of peroxidase isoenzymes shows that two new isoenzymes a and b appeared in hypocotyl cuttings in     

Effect of gamma irradiation on peroxidase isoenzymes during suberization of wounded potato tubers

A comparison of peroxidase isoenzymes in skin, cortex and pith tissues of the potato tuber by thin-layer isoelectric focusing in Sephadex revealed major differences in the isoenzyme patterns. Wounding induced several-fold increases in the peroxidase activity which were correlated with the increased amounts of specific isoenzymes. The anodic and cathodic forms with high activity, normally present in large amounts in skin, were found to be preferentially synthesized in suberizing tissues, suggesting a functional role for peroxidase in the suberization process. Cycloheximide treatment prevented the rapid increase in the content and activity of these specific isoenzymes, which indicated that the increase in peroxidase is due to a de novo synthesis of the enzyme. Suberization is not inhibited by gamma irradiation at sprout-inhibiting dose levels.     

Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Molecular cloning of two novel peroxidases and their response to salt stress and salicylic acid in the living fossil Ginkgo biloba

Background and Aims

Peroxidase isoenzymes play diverse roles in plant physiology, such as lignification and defence against pathogens. The actions and regulation of many peroxidases are not known with much accuracy. A number of studies have reported direct involvement of peroxidase isoenzymes in the oxidation of monolignols, which constitutes the last step in the lignin biosynthesis pathway. However, most of the available data concern only peroxidases and lignins from angiosperms. This study describes the molecular cloning of two novel peroxidases from the ‘living fossil’ Ginkgo biloba and their regulation by salt stress and salicylic acid.

Methods

Suspension cell cultures were used to purify peroxidases and to obtain the cDNAs. Treatments with salicylic acid and sodium chloride were performed and peroxidase activity and gene expression were monitored.

Key Results

A novel peroxidase was purified, which preferentially used p-hydroxycinnamyl alcohols as substrates and was able to form dehydrogenation polymers in vitro from coniferyl and sinapyl alcohols. Two peroxidase full-length cDNAs, GbPrx09 and GbPrx10, were cloned. Both peroxidases showed high similarity to other basic peroxidases with a putative role in cell wall lignification. Both GbPrx09 and GbPrx10 were expressed in leaves and stems of the plant. Sodium chloride enhanced the gene expression of GbPrx09 but repressed GbPrx10, whereas salicylic acid strongly repressed both GbPrx09 and GbPrx10.

Conclusions

Taken together, the data suggest the participation of GbPrx09 and GbPrx10 in the developmental lignification programme of the cell wall. Both peroxidases possess the structural characteristics necessary for sinapyl alcohol oxidation. Moreover, GbPrx09 is also involved in lignification induced by salt stress, while salicylic acid-mediated lignification is not a result of GbPrx09 and GbPrx10 enzymatic activity.     

Characterization of a peroxidase from Sphagnum magellanicum

The peroxidase from Sphagnum magellanicum is strongly inhibited by CN^? and N^3? and is rather heat stable. The pH optimum is 5.0. The peroxidase rapidly degrades the common hydroxycinnamic acids and sphagnum acid to non-phenolic products; t-cinnamic acid itself is not attacked. The significance of these reactions is discussed with respect to earlier investigations on the level of cinnamic acids in Sphagnum magellanicum. The peroxidase consists of five acidic and five basic isoenzymes. This band pattern does not change during the colouring of the moss, so that the peroxidase from the green and the red moss is identical.     

Systematic implications of the peroxidase isoenzymes in the Xanthium strumarium complexes

Starch-slab gel electrophoresis showed two patterns of peroxidase isoenzymes in the polymorphic taxon. Xanthium strumarium L. The “strumarium” morphological complex (X. strumarium, in the sense of Millspaugh and Sherff), the putative indigenous plants of the Old World, contained a different pattern from that shown by putative introductions from the New World. Experimental F₁ hybrids between “strumarium” and other complexes, “italicum-pennsylvanicum”, “chinense”, “oviforme” and “cavanillesii”, had the peroxidase pattern of the American plants. These peroxidase isoenzyme patterns were not influenced by the environmental growing conditions, but, along with partial genetic incompatibility, support the taxonomic separation of X. strumarium from other taxa of section Euxanthium.     

Isoenzymes of cuprozinc superoxide dismutase from Pisum sativum

Two cyanide-sensitive and organic solvent-inactivated superoxide dismutase isoenzymes were purified from pea leaves, Pisum sativum, cv Thomas Laxto     

Purification,characterization and catalytic activity of anionic zucchini peroxidase

The isolation and purification, by preparative electrofocusing, of the major anionic (ZPOA) and cationic (ZPOC) isoenzymes, collected from young zucchini squash, are reported. The M _r and sugar content are similar to those found previously for the major isoenzymes from the ripe fruits and in the range commonly observed for plant peroxidases. The amount of the two cationic enzymes was very low compared with that of anionic ZPOA. The anionic enzyme has been characterized by electronic, circular dichroism, proton NMR and electron paramagnetic resonance spectroscopy. The spectra are qualitatively similar to those of the corresponding anionic horseradish peroxidase (HRPA) derivatives, with minor differences attributable to the particular protein environment around the heme. The kinetics of the enzymatic oxidation of a series of phenols by H₂O₂ have been studied. ZPOA shows a parallel behavior to HRPA, but it is systematically more active than HRPA, indicating that the zucchini enzymes have a marked tendency to carry out oxidation of this type of compounds.     

A phytochemical investigation of Citrus halimii

Essential leaf oils, isoenzymes, protein and polyphenol oxidase-catalyzed browning patterns of Citrus halimii (Rutaceae) are investigated and compared with those of other species of Citrus in order to support the species standing of the recently described C. halimii.     

Treatment of rice straw with selected Cyathus stercoreus strains to improve enzymatic saccharification

Seventeen Cyathus stercoreus isolates were tested for their ability to treat rice straw for improved enzymatic saccharification. These isolates showed a negative correlation between cellulase and xylanase activity and enzymatic saccharification yields. Incubation of rice straw pretreated at 60 °C for 15 min with strain C. stercoreus TY-2 for 25 days resulted in an enzymatic saccharification yield of 57% as compared to a yield of 11% for the same straw in the absence of the fungus. These findings highlight the potential of this isolate for biological pretreatment of rice straw under conditions of low energy input.     

黄孢原毛平革菌合成木素过氧化物酶的营养调控

本文研究了营养条件对黄孢原毛平革茵(Phanerochale chrysosporium)ME-116合成木素过氧化物酶及其同工酶组分的影响.在最适培养条件下获得1500U/L的酶活.高效液相色谱分离的5个同工酶组分中以P_2组分含量最高.低碳高氮培养基最适于酶的合成.降低氮和KH_2PO_4含量致使各组分含量下降,而改变MgSO_4和CaCl_2浓度对P_2组分无影响.表面活性剂吐温80主要通过提高细胞膜透性而增加酶的合成.黎芦醇对5种同工酶组分的合成均有诱导作用.培养基中各营养因子对木素过氧化物酶的合成存在着复杂的交互作用.     

Does a terminal drought tolerance QTL contribute to differences in ROS scavenging enzymes and photosynthetic pigments in pearl millet exposed to drought?

The control of reactive oxygen species (ROS) and the stability of photosynthetic pigments under stress conditions are hypothesized to contribute to drought tolerance. Here we studied how ascorbic peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) isozyme activities and chlorophyll a, b (Chl a, b) and carotenoids (Car) contents responded to water stress and whether they related to presence of a terminal drought tolerance QTL in pearl millet. We used PRLT2/89-33 (QTL donor), H77/833-2 (sensitive), and near-isogenic lines (QTL-NILs) introgressed with the QTL in H77/833-2 background. Under water stress there was no significant change in the total APX activity; only the proportional APX5 activity increased, with higher band intensity in tolerant genotypes. There were no significant changes in total activities of CAT and SOD under water stress, with similar band intensities in all genotypes, and a new CAT isozyme was induced in all genotypes. The photosynthetic pigment content decreased under water stress, although not differently in any genotype. Under water stress, the activities of most APX, CAT and SOD isozymes were closely related to the total chlorophyll/carotenoids ratio. Overall, besides APX5, water stress did not lead to major changes in the profile of isoenzymes involved in ROS scavenging. Similarly, the pigment content under stress did not discriminate genotypes according to the presence/absence of the QTL. This absence of discrimination for the ROS scavenging enzymes and for the pigment content under stress suggests that these traits may not play a key role in terminal drought tolerance in pearl millet.     

Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Immunochemical comparison of the tyrosinase isoenzymes in some Basidiomycetes

The tyrosinase isoenzymes of six agaric species of Basidiomycetes were separated immunochemically by the agar double-diffusion technique and identified using dopa as substrate. The number of isoenzymes identified varied from ten in Agaricus hortensis to one in Flamula alnicola. The isoenzymes in the other species were identical serologically to corresponding isoenzyme components in the A. hortensis complex, The technique provides a relatively simple means for the analytical comparison of tyrosinase isoenzymes from different organisms.     

Molecular weight differences among potato peroxidases

The isoenzyme of potato peroxidase A5 has MW 105 000; C3—94 000; C4 and C5—56 500 C6—48 500. The isoenzymes retain activity on SDS-gels thereby allowing direct measurement of monomeric MW, even in crude extracts. One of the isoperoxidases showed anomalous behaviour on SDS-electrophoresis.     

Characterization of potato (Solanum tuberosum) lactate dehydrogenase isoenzymes by affinity chromatography and hybridization

The five isoenzymes of potato (Solanum tuberasum) lactate dehydrogenase have been resolved by affinity chromatography. Mixtures of isoenzymes LDH-1 and LDH-5 dissociate and reassociate during freezing and thawing to produce five isoenzymes. These results indicate that potato lactate dehydrogenase isoenzymes are primary isoenzymes of the vertebrate type, which are composed of two subunit types.     

Persistence of differences between peroxidase isoenzymes of flax genotrophs in tissue culture

Anionic peroxidase isoenzymes from seedling root, hypocotyl and cotyledon regions of the large (L) and small (S) flax genotrophs were separated on acrylamide gels. Tissue cultures were initiated from each of these regions of the seedlings, and maintained for a 200-day period with six transfers. The differences in electrophoretic mobility of the peroxidase isoenzymes between L and S noted in seedlings, and also in main stems of adult plants, were still present in the tissue cultures.