Extracellular Oxidase Research Articles

О СУБСТРАТНОЙ СПЕЦИФИЧНОСТИ И НЕКОТОРЫХ СВОЙСТВАХ ВНЕКЛЕТОЧНОЙ ОКСИДАЗЫ ИЗ БАЗИДИОМИЦЕТА NEONOTHOPANUS NAMBI

An extracellular enzyme with oxidase activity was isolated from the mycelium of the higher fungus Neonothopanus nambi by mild treatment of the biomass with β-glucosidase. A substrate specificity and some properties of the isolated extracellular oxidase were studied in the present work. Experiments revealed that the extracellular oxidase exhibited activity with most phenolic compounds chosen as model substrates. It is important to note that the enzyme exhibited a catalytic function in the reactions without the addition of exogenous hydrogen peroxide and other mediators. The highest catalytic activity of the enzyme was observed with veratryl alcohol and dihydric phenols, hydroquinone and guaiacol. The enzyme showed lower activity with aromatic azo compounds (ABTS, DAB, o-dianisidine). In reactions with dihydric phenol resorcinol and monophenol, the enzyme efficiency was extremely low. The kinetic parameters of the enzymatic reactions with actively oxidized substrates were determined. The addition of a divalent metal ion chelator (EDTA) did not affect the activity of the enzyme, while the addition of the SH reagent (DTT) increased the catalytic efficiency of the studied oxidase. The totality of the data obtained indicates that the extracellular oxidase of the N. nambi fungus catalyzes the oxidation of a wide range of aromatic compounds under slightly acidic and neutral conditions without the addition of additional mediators (in particular, hydrogen peroxide). This creates the prerequisites for studying the applicability of the enzyme in biomedical analytics.

HISTORICAL HERITAGE IN ANTARCTICA, THE CASA MONETA MUSEUM: A FIRST APPROACH TO THE CHARACTERIZATION OF XYLOPHAGOUS FUNGI AND THEIR POTENTIAL ROLE IN WOOD DAMAGE

The Casa Moneta Museum is located at the base located at an isthmus located on Laurie Island in the South Orkney Islands archipelago. Currently, the museum shows signs of deterioration of the wood (discoloration, fibrous appearance and presence of mycelium) and it is necessary to characterize this mycobiota to analyze the potential role in the biodegradation process. An assessment of the potential risk associated with fungal communities could provide valuable information for taking conservation action at the Casa Moneta Museum. The aim of this work was to evaluate the extracellular oxidase production of fungi recovered from deteriorated wood at the museum and to analyze the biodegradation risk index of three sampled sites in the building. The enzymatic profile showed the potential degradation of Cadophora spp. and Tulasnella albida strains. The analysis performed integrated data on several parameters like occurrences, species diversity, dominant species, wind direction, relative humidity, growth temperature, type of dispersion propagules and substrate, indicating Site 2 has a major biodegradation risk index which suggests that this site should be the immediate target for conservation efforts.

Construction of the SHP-GLOX lignin regulation system and its application in rice straw

BackgroundThere is great productivity of rice(Oryza sativa L. spp. japonica) straw in China, which is a potential source of biomass for biofuel and forage. However, the high levels of lignins in rice straw limited its usage and induced the formation of agricultural waste. In order to modify the lignins contents to improve biofuel production and forage digestibility, we selected Soybean hull peroxidase (SHP) and Glyoxal oxidase (GLOX) as candidate genes to improve quality of rice straw. SHP, a class III plant peroxidase, is derived from multiple sources. It has several advantages, such as high resistance to heat, high stability under acidic and alkaline conditions, and a broad substrate range. SHP is speculated to be useful for lignin degradation. Glyoxal oxidase (GLOX) is an extracellular oxidase that can oxidize glyoxal and methylglyoxal in the extracellular medium to generate H2O2.ResultsIn the present study, the SHP and GLOX genes in pCAMBIA3301-glycine-rich protein (GRP)-SHP-GLOX, designated the K167 vector, were optimized and introduced into rice embryos using Agrobacterium-mediated transformation. Positive transgenic rice embryos were examined using molecular, physiological, biochemical and fermentation tests. The outcomes suggested that SHP degraded lignin effectively.ConclusionsThis research has created a rice breeding material with normal growth and yield but stalks that are more amenable to degradation in the later stage for use in breeding rice varieties whose stalks are easily used for energy. Our results will improve the industrial and commercial applications of rice straw.

Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.

Extracellular Oxidase from the Neonothopanus nambi Fungus as a Promising Enzyme for Analytical Applications.

The extracellular enzyme with oxidase function was extracted from the Neonothopanus nambi luminescent fungus by using mild processing of mycelium with β-glucosidase and then isolated by gel-filtration chromatography. The extracted enzyme is found to be a FAD-containing protein, catalyzing phenol co-oxidation with 4-aminoantipyrine without addition of H2O2, which distinguishes it from peroxidases. This fact allowed us to assume that this enzyme may be a mixed-function oxidase. According to gel-filtration chromatography and SDS-PAGE, the oxidase has molecular weight of 60kDa. The enzyme exhibits maximum activity at 55-70°C and pH 5.0. Kinetic parameters Km and Vmax of the oxidase for phenol were 0.21mM and 0.40µMmin-1. We suggest that the extracted enzyme can be useful to develop a simplified biosensor for colorimetric detection of phenol in aqueous media, which does not require using hydrogen peroxide.

Acido-thermotolerant fungi from Boiling Springs Lake, LVNP: Potential for lignocellulosic biofuels

Acidic geothermal environments such as those in Lassen Volcanic National Park (LVNP) may provide novel organisms and enzymes for conversion of plant lignocellulose into ethanol, a process that typically requires hot and acidic pre-treatment conditions to hydrolyze cell wall polysaccharides to fermentable sugars. We evaluated seven Ascomycete fungi associated with LVNP’s Boiling Springs Lake (BSL) for utilization of lignocellulose material. We screened the fungi for growth pH and temperature optima, and for growth on purified or natural plant cell wall components. We also examined potential lignin degradation using a (per)oxidase assay, and screened for the presence of potential (hemi)cellulose degradation genes with PCR. Growth analysis showed Acidomyces and Ochroconis grew best at 35–45 °C and pH < 4, and grew up to 48–53 °C. In contrast, Aspergillus , Paecilomyces , and Penicillium preferred cooler temperatures for acidic media (25–35 °C), but grew up to 48 °C. Phialophora only grew up to 27 °C under both acidic and neutral conditions, and Cladosporium showed a preference for cool, neutral conditions. The most promising material utilizers, Acidomyces , Ochroconis , and Paecilomyces , used cellobiose and xylan, as well as pine and incense cedar needles, for growth at 40 °C and pH 2. Acidomyces and Ochroconis showed extracellular (per)oxidase activity at 40 °C and pH 2, and PCR screening showed Acidomyces , Paecilomyces , and Ochroconis contain orthologs to known fungal lignocellulose degradation genes, including glucanases and xylanases. We conclude that the BSL-adapted taxa Acidomyces , Ochroconis , and Paecilomyces may be promising sources of enzymes that combine heat- and acid-tolerance, potentially valuable in streamlining the pre-treatment of lignocellulosic biofuels.

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

ABSTRACTOxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U mg−1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6 h. The enzymatic activity was not influenced by metal ions and chemical agents (K+, Na+, Zn2+, Fe3+, Mn2+, Mg2+, glucose, urea, lactate) commonly found in serum and urine, with Cu2+ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca2+ and Fe2+. Its Km and Kcat for oxalate were found to be 0.45 mM and 85 s−1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50 mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.

Wood-inhabiting fungi of the Białowieża virgin forest in Poland. XVIII. Amylocystis lapponica (Romell)Bond. &amp; Sing.

The author examined the microstructure of the fruitbody and culture of polypore &lt;i&gt;Amylocystis lapponica&lt;/i&gt; (Romell) Bond. &amp; Sing., common in the Białowieża virgin forest on lying logs of &lt;i&gt;Picea excelsa&lt;/i&gt; with symptoms of advanced brown rot produced by &lt;i&gt;Fomitopsis rosea&lt;/i&gt; (Alb. &amp; Schw. ex Fr.) P. Karst. In the fruitbody a monomitic hyphal system was revealed consisting for the most part of thick-walled nodose-septate hyphae 4-10,5 μm thick. Moreover, by examination of four cultures obtained from basidiospores, it was found (1) that the diploid mycelium gives nearly always a negative reaction, whereas the haploid mycelia give positive reactions in tests for extracellular oxidase, and (2) that the fungus is tetrapolar.

Grzyby zasiedlające drewno w Puszczy Białowieskiej. XVII. Ceriporiopsis gilvescens (Bres.) Domański [Wood-inhabiting fungi in Białowieża virgin forest in Poland. XVII. Ceriporiopsis gilvescens (Bres.) Domański

&lt;em&gt;Ceriporiopsis gilvescens&lt;/em&gt; (Bres.) Domański producing white rot in wood of deciduous trees is the type species of the genus &lt;em&gt;Ceriporiopsis&lt;/em&gt; Domański. In order to complete its generic description, the culture of the fungus was examined and its species code established. Interfertility studies, with single spore cultures isolated from sporophores derived from three sources in Poland have shown that the fungus is heterothallic and has the tetrapolar type of interfertility. It gives also positive reaction in the test for extracellular oxidase In this connection (1) these characters have been introduced in the description of !the genus &lt;em&gt;Ceriporiopsis&lt;/em&gt;, and (2) the species &lt;em&gt;Poria placenta&lt;/em&gt; (Fr.) Cke sensu J. Erikss. is excluded from this genus

Microbial Community Analysis in Bio-Filter Bed of Iron and Manganese Removal Treating High Iron, Manganese and Ammonia Nitrogen Groundwater

The integrated process of spray aeration, falling water aeration and bio-filter bed was used for treating groundwater with high iron, manganese and ammonia nitrogen (NH4+-N). The removal efficiencies of iron, manganese and NH4+-N reached 99.1%, 95.0% and 85.2%, and corresponding effluent iron and manganese decreased to 0.1 and 0.05 mg/L. Microbial analysis results indicated that theβ-Proteobacteriawas predominant microorganisms, in whichGallionellaandLeptothrixwere main iron-removal bacteria and manganese-removal bacteria, respectively. Simultaneously,Pseudomona, belonged toγ-Proteobacteria, could absorb and oxidize free manganese to be manganese dioxide (MnO2) by extracellular oxidase.

Expression of LOX is upregulated under hypoxia in metastatic colorectal carcinoma cells

Lysyl oxidase (LOX) is an extracellular amine oxidase catalyzing the formation of lysine-derived cross-linkages in collagen and elastin in the extracellular matrices. Four human paralogs of LOX (LOXL1, LOXL2, LOXL3, and LOXL4) have been identified, each encoding the functional domains required for the amine oxidase activity of LOX. Recently, upregulation of the LOX and LOXL2 expression was reported to be significantly correlated with absence of lymphovascular invasion in tumor tissues from colorectal adenocarcinoma patients, suggesting that the oxygen tension around the tumors may be an important factor for expressional regulation of these genes in colorectal carcinomas. To evaluate the effects of hypoxia on expression of LOX and LOXL2 in colorectal carcinomas, we performed promoter assays and RT-PCR analysis under hypoxia in colorectal carcinoma cell lines, HT-29 and HCT-116. LOX expression was upregulated under hypoxia in both HT-29 and HCT-116 cells, whereas LOXL2 expression was not affected by hypoxia in those cells. In the highly metastatic HCT-116 cells, LOX showed a higher level of upregulation than in HT-29 cells, suggesting that LOX upregulation may be associated with increased invasiveness and metastatic potential in colorectal carcinomas.

Isolation of thermophilic fungal strains producing oxido-reductase and hydrolase enzymes from various Tunisian biotopes

The present study surveys 37 thermophilic fungal strains, that were isolated from various Tunisian biotopes such as litter, husk and compost, for their involvement in lignin degradation activities. Oxido-reductase and hydrolase activities were screened. Oxido-reductase activities, such as Extracellular Oxidase, Laccase, Tyrosinase and Peroxidase, were tested. Hydrolase activities, namely Lipase, Protease, Amylase and Cellulase, were also revealed. Strain ST26, isolated from locally made compost, and showed an important laccase activity with ABTS and DMP, was selected. Laccase activity of strain ST26 was 1575 U/l in liquid media. This strain was identified and was belonging to Scytalidium genus and more precisely to the specie Scytalidium thermophilum using 5.8S rRNA gene sequencing analysis.

Extracellular ATP and NADPH oxidase regulate plasma membrane calcium channels

Extracellular ATP and NADPH oxidase regulate plasma membrane calcium channels

The dynamics of oxidase activity during cultivation of basidiomycetes from the genus Trametes Fr.

Twenty strains of the wood-degrading fungi from the genus Trametes Fr., capable of synthesizing laccases, were screened according to the changes in the oxidase activity in a submerged culture. The range of maximal efficiency of various species with respect to extracellular oxidase activity was determined. The absence of correlation between the oxidase activity in a submerged culture and the size of colored zone on agar media (Bavendamm reaction) was demonstrated. The most efficient strains, T. hirsita 56 and T. ochracea 92-78, were used to produce laccases, homogeneous according to SDS electrophoresis data. A number of biochemical parameters characteristic of these enzymes were determined.

Evidence for an extracellular l–amino acid oxidase in nitrogen-deprived Phaeodactylum tricornutum (Bacillariophyceae) and inhibition of enzyme activity by dissolved inorganic carbon

T.A.V. Rees and V.J. Allison. 2006. Evidence for an extracellular l–amino acid oxidase in nitrogen-deprived Phaeodactylum tricornutum (Bacillariophyceae) and inhibition of enzyme activity by dissolved inorganic carbon. Phycologia 45: 337–342. DOI: 10.2216/04-92.1Nitrogen-deprived cells of the marine diatom Phaeodactylum tricornutum possessed extracellular l–amino acid oxidase (EC 1.4.3.2) activity. No enzyme activity was detectable in nitrogen-replete cells. When nitrogen-deprived cells were provided with 100 μM alanine, the amino acid was metabolised, with both pyruvate and H2O2 accumulating stoichiometrically in the medium; this accumulation was largely prevented if the cells were preincubated with the membrane-impermeable protease Proteinase K. Following the transfer of nitrogen-replete cells to nitrogen-free medium, development of enzyme activity lagged, with little or no enzyme activity detectable until 4 h after the onset of nitrogen deprivation. This lag was followed by rapid development of enzyme activity during the ensuing 20 h. In addition to alanine, methionine, methionine sulfoximine, leucine, glutamate and valine, at the same initial concentration of 100 μM, were exclusively metabolised by an amino acid oxidase. Histidine was partially metabolised by an amino acid oxidase, whereas arginine and lysine were transported into the cell with little or no metabolism by an amino acid oxidase. Neither serine nor proline was used. In general, the neutral nonpolar and acidic polar amino acids were used via an extracellular amino acid oxidase, whereas the basic polar amino acids were transported. l–Amino acid oxidase activity was inhibited in darkness and in light in the presence of the photosynthetic inhibitor DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea). In addition, enzyme activity decreased on addition of bicarbonate to cells in light. Together these observations suggest that extracellular l–amino acid oxidase activity was inhibited by dissolved inorganic carbon.

Properties of and Prospects for Practical Use of Extracellular L-Glutamate Oxidase of Streptomyces sp. Z-11-6

The properties of extracellular L-glutamate oxidase, isolated and purified from Streptomyces sp. Z-11-6 (specific activity, 50.8 U/mg protein; yield, 40%), were studied. A photometrical method of determination of activities of alanine- and aspartate aminotransferases, based on the use of the L-glutamate oxidase and peroxidase, has been developed. This method is sufficiently sensitive to be used for the determination of aminotransferase activities in biological fluids. The presence of other amino acids did not interfere with the analysis and had no effect on the results of determination.

Growth of wood-inhabiting Lentinus species from Cameroon in laboratory culture

Three species of Lentinus from Cameroon, L. sajor-caju (Fr.) Fr., L. squarrosulus Mont. and L. velutinus Fr. were investigated in laboratory culture. L. velutinus produced xylarioid outgrowths in both monosperm and polysperm cultures, but these structures were observed only to a lesser extent in polysperm cultures of L. squarrosulus , and those of L. sajor-caju , as well as their respective monosperm cultures, remained sterile. Extracellular oxidase tests revealed all three species to be white-rot fungi.

Characterization of an extracellular salicyl alcohol oxidase from larval defensive secretions of Chrysomela populi and Phratora vitellinae (Chrysomelina)

Larvae of a number of chrysomelid leaf beetles sequester phenol glucosides such as salicin from their food plants, i.e. Salix and Populus spp. Salicin is hydrolyzed in the glandular reservoir of the defensive glands. The resulting salicyl alcohol (saligenin) is oxidized by an extracellular oxidase. The product salicylaldehyde accumulates as major defensive compound. The secretions from Chrysomela populi and Phratora vitellinae were preserved in saturated ammonium sulfate solution and subjected to micro-purification of the oxidase by means of electrophoretic methods. The enzyme from P. vitellinae has a native M r of 334 000 and a subunit M r of 79 000 indicating a tetrameric enzyme. The isoelectric points of the enzymes from C. populi and P. vitellinae are at pH 5.4 and 5.2, respectively. In the oxidation of salicyl alcohol oxygen functions as electron acceptor yielding hydrogen peroxide as product. Hydrogen peroxide does not accumulate in native secretions but appears to be degraded most likely by a catalase. The oxidases from the two species show broad pH optima in the range 5.5 to 6.5, they oxidize salicyl alcohol as main substrate. Minor substrates are several ortho-substituted and to a lesser extent meta- but not para-substituted benzyl alcohols. In the presence of 8-hydroxygeraniol only trace amounts of the respective aldehyde are formed. The K m values of salicyl alcohol are 132 mM ( C. populi) and 63 mM ( P. vitellinae). The extracellular enzyme, which is functionally related to fungal aryl alcohol oxidase (EC 1.1.3.7) and vanillyl alcohol oxidase (EC 1.1.3.38) was named salicyl alcohol oxidase. The continuous formation of salicylaldehyde in the glandular reservoir can be compared to the operation of an enzyme reactor. Due to its low aqueous solubility the produced aldehyde steadily leaves the aqueous reaction fluid and builds up an organic phase which may account for 15% of the total liquid volume of the secretion.

Streptomyces sp. Z-11-6, a novel producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254 Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep-mutagenesis and selection procedure allowed a genetically stable strain, Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.

Extracellular L-glutamate oxidase ofStreptomyces sp. Z-11-6: Obtainment and properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain,Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6–1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase fromStreptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1–0.5% promoted the secretion of the extracellular glutamate oxidase.