Ligninolytic enzyme complex ofArmillaria spp

Ten strains belonging to five species of European Armillaria (Fr.:Fr.) Staude were examined for their ability to produce laccase, lignin peroxidase, manganese-dependent peroxidase and manganese-independent peroxidase. No lignin peroxidase activity was observed in any of the strains. Manganese-dependent peroxidase production by all tested strains was low. Difference in the ratio of laccase to manganese-independent peroxidase in strains of A. gallica in comparison to all other species was detected.     

Comparative studies of lignin peroxidases and manganese-dependent peroxidases produced by selected white rot fungi in solid media

Abstract The effect of various incubation conditions and media composition on ligninolytic activity by selected strains of white-rot fungi was determined in solid media. When compared to conventional methods using liquid media or woody substrates, this method is fast, simple and also quantitative. Manganese-dependent peroxidase was easily detected in all strains studied. However, detection of lignin peroxidase required optimisation of both growth medium and enzyme assay conditions. Using this method, we showed that the role of nitrogen and oxygen in ligninolytic activity varies and that conditions must be optimised for each individual even within the same species. Furthermore, several white rot fungi produced manganesedependent peroxidase during the primary growth phase. Keywords: Manganese-dependent peroxidase; Lignin peroxidase; White rot fungus     

Flow-injection binding assays: a way to increase the speed in binding analyses

A flow-injection analysis system was equipped with a small column containing immobilized concanavalin A. Pulses containing glucosides or glycoproteins were passed over the column, the lectin bound the carbohydrates. By using horseradish peroxidase as a labeled carbohydrate and letting it compete with other glucosides or mannosides a competitive binding assay for the latter was set up. When the enzyme activity had been evaluated, the column was rinsed and reconditioned, allowing a new assay to be run. To speed up the assay, substrates for the enzyme marker, peroxidase, were present in the perfusing buffer. A computerized evaluation of the absorbance peak allowed the time of the assay cycle to be reduced to 70 s. The sensitivity of this binding assay was fully comparable with those reported for other systems using the same reactants.     

A simple method for determining the relative activities of individual peroxidase isozymes in a tissue extract

A peroxidase specific zymogram staining procedure has been designed which not only locates peroxidase isozymes which have been resolved electrophoretically, but simultaneously estimates the relative activity of each peroxidase isozyme in the system under study. This quantitative assay of individual peroxidase isozymes is chronometric; the time required for the appearance of a peroxidase band is inversely proportional to its activity. By recording the time required for the appearance of each peroxidase isozyme in a tissue extract, the percent contribution each isozyme makes toward the total peroxidatic activity of that tissue can readily be determined.     

A Rapid Assay for Monitoring Peroxidase Activity in Melon as a Marker for Resistance to Pseudoperonospora cubensis

Abstract A rapid assay for monitoring peroxidase activity in melon as a marker for resistance to Pseudoperonospora cubensis was developed. A high correlation was demonstrated between peroxidase activity and resistance in test wells containing leaf disks of melon plants with known levels of susceptibility or resistance to P. cubensis. The possibility of using such an assay for a preliminary selection of melons resistant to P. cubensis is suggested.     

An improved assay method for thyroid peroxidase applicable for a few milligrams of abnormal human thyroid tissues

A peroxidase assay method (Mini assay method) which is applicable for a minute amount (as small as a few mg) of thyroid tissue was developed, employing guaiacol or iodide as the second substrate. This method is a modification of the previous one (Ordinary assay method): the volume of the reaction mixture was reduced to about one-tenth with prior solubilization of the enzyme. The correlation between the Mini assay and Ordinary assay methods, and between the guaiacol and iodide assays by both methods were satisfactorily good, but the iodine content of thyroglobulin was found to be not directly correlated to the peroxidase activities. Protein-based specific activities of peroxidase from normal human thyroid tissue were about 0.030 guaiacol units/mg protein and 0.0066 iodide units/mg protein, which were slightly higher than those of porcine thyroid tissue. The Mini assay method developed in the present study was used for the determination of peroxidase activity in a small amount (1-8 mg) of thyroid tissue obtained by means of a needle biopsy from patients with thyroid disorders. One specimen (goitrous cretinism) showed no peroxidase activity in both the guaiacol and iodide assays, and three specimens (two chronic thyroiditis, one familial nontoxic goiter) possessed no ability to catalyze the oxidation of iodide in spite of the high reactivity towards guaiacol, suggesting the presence of an abnormal peroxidase in these tissues.     

Peroxidases from marine microalgae

Peroxidase activity was detected in cell-free extractsof strains of three species of the marine microalgae,Porphyridium purpureum, Phaeodactylumtricornutum and Dunaliella tertiolecta. However, no bromo- or chloroperoxidase activity wasdetected in any, using the standard 2-chlorodimedoneassay. Only the extract from P. purpureumoxidized iodide and this peroxidase was partiallypurified via anion-exchange chromatography. KI ando-dianisidine assay of the fractions indicatedthat only one peroxidase was present. Characterization of the thermally labile enzymesuggested that it is a heme-containing peroxidase,with a molecular weight of approximately 36,000.     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms

Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity.     

Industrial Dye Decolorization by Laccases from Ligninolytic Fungi

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized. Received: 26 May 1998 / Accepted: 7 August 1998     

Characterization of the hydroperoxide-reducing activity of human plasma

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.     

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.     

A sensitive enzyme immunoassay for human basic fibroblast growth factor

A sensitive sandwich enzyme immunoassay for human basic fibroblast growth factor (HbFGF) was developed employing three monoclonal antibodies (MAb3H3, MAb98 and MAb52). The Fab' fragment of MAb3H3 which inhibits HbFGF biological activity was conjugated to horseradish peroxidase. A mixture of MAb52 and MAb98 was used in the solid phase. Neither human acidic fibroblast growth factor, hst-1/KS3 product nor acid denatured HbFGF was cross-reactive in this assay system. The detection limit of this assay system was 1 pg/well. Using this assay, some tumor cell lines were revealed to produce a higher level of bFGF than a normal one. Serum samples from normal volunteers were also assayed, and immuno-reactive HbFGF could be detected in 16 out of 57 samples at range 30 approximately 206 pg/ml.     

Decolorization of triphenylmethane dyes by wild mushrooms

Triphenylmethane dyes such as Crystal Violet (CV) and Malachite Green (MG) are common textile dyes. MG, which is toxic to humans, is widely used in aquaculture as an antifungal agent. In this study, 56 mushroom strains from 12 species of wild mushrooms were examined on dye-containing PDA plates to evaluate their potential for the bioremediation of synthetic dyes. Pycnoporus coccineus, Coriolus versicolor, and Lentinula edodes showed fair growth on CV, but only a few survived on MG. However, a decolorization experiment in an aqueous system revealed that the growth on MG-containing solid medium did not directly match the decolorization of MG in the aqueous system. C. versicolor IUM0061 grew well on both MG and CV plates, but could not decolorize MG in the reaction mixture. Conversely, HPLC analysis revealed that P. coccineus IUM0032, which could not grow on the MG plate, completely mineralized MG within 3 days. A subsequent enzyme activity assay revealed a high lignin peroxidase activity in the reaction mixture, indicating that lignin peroxidase is the key enzyme involved in degradation of MG in P. coccineus IUM0032.     

Heat-stability of peroxidase in mycelia of some toxigenic and nontoxigenic Aspergilli and Penicillia

Seven-day-old mycelia from 19 cultures of Aspergillus and 12 cultures of Penicillium were heated to 50, 60, 65, 70, 75, 80, 85, 90 or 95 C for no more than min, and tested for residual peroxidase. The peroxidase from all aspergilli survived heating at 50 through 80 C. Peroxidase from toxigenic strains of Aspergillus flavus, Aspergillus parasiticus and Aspergillus ochraceus survived heating at 85 C and often at 90 C, whereas peroxidase from nontoxigenic strains of A. flavus was inactivated at 90 C and markedly reduced in activity at 85 C. Peroxidase from all penicillia survived heating at all temperatures through 80 C, although the activity of several cultures was reduced at 80 C. Peroxidase activity in mycelia of two strains of Penicillium cyclopium and one of Penicillium puberulum failed to survive heating at 85 C. One strain each of Penicillium roqueforti and Penicillium viridicatum exhibited some peroxidase activity after heating at 90 C, whereas the peroxidase of all other penicillia was inactivated at this temperature.     

A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.

A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.     

Biochemical activity of opportunistic antibiotic and chloramine-sensitive and resistant microorganisms isolated from healthy and sick people

Activity of oxidation-reduction enzymes such as succinate dehydrogenase, peroxidase and catalase was studied in staphylococci isolated from healthy persons and patients as well as from the air and implements of medical institutions. The isolates were resistant either to antibiotics or to chloramine B or to the both. The results showed that development of resistance to antibiotics and chloramine B in the staphylococci was accompanied by a decrease in the activity of succinate dehydrogenase, peroxidase and catalase. In the strains resistant only to chloramine B the activity of the enzymes was practically at the same level as in the strains resistant only to antibiotics. In the strains resistant to both antibiotics and chloramine B, the activity of succinate dehydrogenase, peroxidase and catalase did not practically differ from that in the strains resistant either to antibiotics or to chloramine B.     

A new sensitive colorimetric assay for peroxidase using 3,3'-diaminobenzidine as hydrogen donor

A new simple colorimetric assay for measurement of peroxidase activity using 3,3′-diaminobenzidine tetrahydrochloride as hydrogen donor is described. The DAB is stable under the usual assay conditions, and its rate of auto-oxidation is negligible. Under optimal conditions, a linear relationship is found between peroxidase concentration and the rate of oxidation of 3,3′-diaminobenzidine tetrahydrochloride (ΔA_405nm/min). Using horseradish peroxidase, the DAB method appears more sensitive than the o-dianisidine and the guaiacol assays for peroxidase. This method can also be used for measurement of peroxidase activity in tissue fractions.     

Vanadium effect on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase in vitro.

The effect of vanadium (V) on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase has been studied. A competitive inhibition pattern was evident for vanadate ions on the activity of horseradish peroxidase (Ki = 41.2 microM). No significant inhibitory effects were found when V(V) was tested with catalase and when either V(IV) or V(V) were assayed with glutathione peroxidase. For the latter, the effect of V on the different components of the reaction system was investigated. V(V) did not significantly affect SOD activity when assayed with the sulfite method, which is devoid of interferences with V(V); however, there was an apparent inhibitory dose-response pattern for either V(IV) or V(V) using the pyrogallol assay, owing to an interference of pyrogallol with the metal. Besides, no significant binding of V(IV) or V(V) to the enzyme could be demonstrated. The lack of a direct inhibitory effect of V on the activity of the main antioxidant enzymes suggests that many biological and toxicological effects of V may be mediated more by oxidative reactions of the metal or of its complexes with physiologically relevant biomolecules than by a direct modulation of enzymatic activities.     

Superoxide dismutase,catalase, and glutathione peroxidase in the swim bladder of the physoclistous fish,Opsanus tau L

Summary The antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase were measured in the rete mirabile and gas gland epithelium area of the swim bladder of the toadfish Opsanus tau. When the concentration of enzyme in the swim bladder was compared with the concentration in other organs (kidney, heart, gills) of the same fish, the swim bladder was found to have the highest concentration of superoxide dismutase but relatively low levels of glutathione peroxidase and catalase.Cytochemical assay for the peroxidatic activity of catalase confirmed that virtually no catalase is present in epithelial cells of the gas gland. A similar assay for peroxidase revealed a cyanide-sensitive peroxidase in the multilamellar bodies of these cells. Most of the catalase and peroxidase in the rete mirabile appears to be confined to the granules of neutrophils and the cytoplasm of erythrocytes. Enzyme activity in the neutrophils is not inhibited by 10^-1 M KCN. Cyanide does appear to inhibit the peroxidase activity in erythrocytes but has little effect on catalase in these cells.Supported by grant No. HL23338 from the National Institutes of Health