Phenoloxidase Production Research Articles

Lignocellulosic supplementation of the culture medium for mycelial development and production of phenoloxidase enzymes from Amazonian strains of gasteroid fungi (Basidiomycota)

Strains of gasteroid fungi are capable of oxidizing phenolic components and producing enzymes of great commercial value. However, the difficulty in producing biomass and, consequently, the low levels of expression of enzymes produced by these fungi hinder the advancement of biotechnological studies. A possible alternative may be supplementing the culture medium with lignocellulosic substrates, as observed in the cultivation of edible mushrooms, for example. Thus, the objective of this study was to analyze the effect of supplementing a potato-based culture medium with different concentrations of wheat and rice bran on mycelial development and the production of phenoloxidase enzymes of Amazonian strains of Cyathus and Geastrum. The basidiomas were collected in the Amazon of Pará, and the strains were obtained from mycelial isolation on Potato Dextrose Agar (PDA) medium. The strains were inoculated in semi-solid and liquid potato-based medium containing 10% and 20% rice bran and 10% and 20% wheat bran. For a period of 20 days, mycelial growth was observed in solid form, and for 40 days, the development of the strains in liquid medium was monitored, all incubated at 25°C in the dark. Growth and production of fresh and dry biomass were measured, as well as the production of phenoloxidase, arising from growth in liquid medium. Supplementation using 10% wheat bran was the most promising for the radial growth of four of the six species tested (C. limbatus, G. hirsutum, G. echinulatum, and G. schweinitzii), in addition to being the best treatment for the production of biomass and phenoloxidase enzymes for all species. Considering that wheat bran is easily accessible, this study stimulates basic science, especially in the Amazon, where Funga is still underestimated in terms of diversity and biotechnological potential.

Silver nanoparticles loaded pyrrole based pesticidal metabolites (AgNps-PFM) nanoconjugate induced impact on the gut microbion and immune response against lepidopteron pest Spodoptera litura (Fab.)

Pest control measures using nanobiotechnology principles is now being extensively utilized in the various parts of the world. Understanding the molecular mechanism behind the pestiicdal activity of the nanomaterials is essential to exploit the principles of nanomaterials as an effective pest control strategy. In the present investigation, impact of the nanoconjugate prepared from biogenic silver nanoparticles-fungal based insecticidal metabolites (AgNps-FM) on the gut microbion and immune response against major lepidopteron pest Spodoptera litura (Fab.) was studied. Nanoconjugate was prepared from silver nanoparticles from Pomegranate (Punica granatum) peel extract with the fungal based pesticidal metabolites. Highly stable nano structural nano conjugate thus obtained was evaluated against gut microbion and immune response of Spodoptera litura. Gut microbion status was studied by determination of microbial count, extra cellular enzymes potential of the gut microbial isolates adopting culture dependent methods. Nanoconjugate induced effect on the immune response was done by recording total hemocyte count and reduced total haemocyte count, phenol oxidase activity and lectin expression pattern Synthesized nanoconjugate brought about notable impact on the gut microbion count and their extra cellular enzyme potential. Reduced haemocytes count, poor lectin expression and reduced phenol oxidase production reveals the immune suppressive activity. High efficacy of the Nanoconjugate thus obtained would suggest the possible utilization of Nanoconjugate as an effective, safe pesticidal agent against economic important insect pests.

Soil respiration and extracellular enzyme production respond differently across seasons to elevated temperatures

The activity of extracellular enzymes is one control on soil organic matter decomposition and serves as a driver of heterotrophic soil respiration. To understand how temperature sensitive enzyme reactions will influence the processing of soil carbon substrates at elevated temperatures, we deployed a passive warming experiment in a deciduous forest with highly invaded understory plant communities and high soil nitrogen concentrations. We seasonally assessed six extracellular enzyme activities and measured soil respiration in the field and in laboratory incubations, to determine if soil carbon use and nutrient cycling responded to 3.5 years of warming. Field measurements indicate soil respiration was 24% lower and the production of the recalcitrant C-acquiring enzyme phenoloxidase was also lower in warmed plots during fall and winter. In the spring, phosphate (P) acquiring enzyme activity increased in response to warming. In lab incubations, soil respiration was not different between warmed and control soils. Despite minimal changes to C stores after 3.5 years of warming, recalcitrant C-use and nutrient processing enzymes responded differentially to higher temperatures in fall, winter, and spring. This suggests that C- and nutrient cycle responses to warming can change throughout the seasons, perhaps mediated by plant phenological changes.

Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated in Italy

BackgroundCryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. The virulence factors involved in cryptococcosis pathogenesis include the presence and the size of the polysaccharide capsule, the production of melanin by phenoloxidase, the growth at 37°C and the enzyme secretion like proteinase, phospholipase and urease. Many other enzymes are secreted by C. neoformans but their role in the fungus virulence is not yet known. AimsIn order to investigate this topic, we compared the phospholipase production between strains from patients and from bird droppings, and we examined its relationship to phenoloxidase production. We further characterized the strains by determining the activity of 19 different extracellular enzymes. MethodsTwo hundred and five Italian C. neoformans clinical isolates and 32 environmental isolates were tested. Phenoloxidase production was determined by the development of brown colonies on Staib's agar. Extracellular phospholipase activity was performed using the semiquantitative egg-yolk plate method. API ZYM commercial kit was used to observe the production and the activity of 19 different extracellular enzymes. ResultsStatistical analysis of the results showed a significantly higher phospholipase activity in the clinical isolates than in the environmental isolates. No significant difference about the phenoloxidase production between both groups was found. Regarding the 19 extracellular enzymes tested using the API ZYM commercial kit, acid phosphatase showed the highest enzymatic activity in both groups. Concerning the enzyme α-glucosidase, the clinical isolates presented a significantly higher positivity percentage than the environmental isolates. A hundred percent positivity in the enzyme leucine arylamidase production was observed in both groups, but the clinical isolates metabolized a significantly greater amount of substrate. ConclusionsThe higher phospholipase production in the clinical isolates group confirms the possible role of this enzyme in the cryptococcosis pathogenesis. The extracellular activities of the enzymes acid phosphatase, α-glucosidase and leucine arylamidase, tested by means of the API ZYM commercial kit, appear to be very interesting. Many studies indicate that these enzymes are involved in the virulence of bacteria and parasites; our results suggest their possible role as virulence factors in Cryptococcus infections too.

Optimum Conditions for Phenol Oxidase (Laccase) Production from Azotobacter chroococcum by Submerged Fermentation

Phenol oxidases (laccase), copper-containing oxidase enzymes, are found in many plants, fungi and microorganisms. Many factors that influence laccase production from Azotobacter chroococcum are determined in this study, these factors are: carbon source, nitrogen source, pH, incubation temperature and incubation period. The results showed that the best carbon and nitrogen sources for laccase production are sucrose and yeast extract respectively, and the best factors for laccase production conditions are pH 7.0 with an incubation period of 6 days at temperature 30 Cᵒ.

Diversity and Distribution of Phenol Oxidase Producing Fungi from Soda Lake and Description of Curvularia lonarensis sp. nov.

Soda lake is hyper alkaline and saline habitat located in closed craters with high evaporation rate. In current study fungal diversity from water and sediment samples of a soda lake (Lonar lake) located in Buldhana district of Maharashtra, India was investigated using extensive culturomics approach and mimicking the natural conditions of Lonar lake in culture media. A total of 104 diverse isolates of extremophilic fungi were recovered from this study and phylogenetically characterized by internal transcribed spacer (ITS) region sequencing. In addition, due to important role of phenol oxidase, and peroxidase in degradation of toxic phenol, lignin, etc., all isolated pure cultures were also screened for extracellular phenol oxidase and peroxidase production potential. Diversity analysis indicated that different groups of extremophilic fungi are present in the water and sediment samples of Lonar lake. A total of 38 species of fungi belonging to 18-different genera were recovered. Out of 104 isolates 32 showed ≤97% sequences similarity, which were morphologically different and could be potential novel isolates of extremophilic fungi. However, out of 104 isolates only 14 showed the extracellular phenol oxidase production potentials at alkaline pH. Curvularia sp. strain MEF018 showed highest phenol oxidase production at alkaline condition and had low sequence similarity with previously characterized species (96% with Curvularia pseudorobusta). Taxonomic characterization (morphological and physiological) and multi locus sequence analysis (MLSA) using combined alignment of ITS-LSU-gpd of strain MEF018 showed that it is a novel species of the genus Curvularia and hence proposed as Curvularia lonarensis sp. nov.

Ant sperm storage organs do not have phenoloxidase constitutive immune activity

The prophenoloxidase system (proPO-AS) is a primordial constituent of insect innate immunity. Its broad action spectrum, rapid response time, and cytotoxic by-products induced by phenoloxidase (PO) production contribute to the effective clearing of invading pathogens. However, such immune reactions may not be optimal for insect organs that evolved to have mutualistic interactions with non-self-cells. Ant queens are long-lived, but only mate early in adult life and store the sperm in a specialized organ, the spermatheca. They never re-mate so their life-time reproductive success is ultimately sperm-limited, which maintains strong selection for high sperm viability before and after storage. The proPO-AS may therefore be inappropriate for the selective clearing of sexually transmitted infections, as it might also target sperm cells that cannot be replaced.We measured PO enzymatic activity in the sperm storage organs of three ant species before and after mating. Our data show that no PO is produced in the sperm storage organs, relative to other somatic tissues as controls, and that these negative results are not due to non-detection in small volumes as non-immune-relevant catalase activity in single spermatheca fluid samples of both virgin and mated queens was significant. The lack of PO activity in sperm storage organs across three different ant species may represent an evolutionarily conserved adaptation to life-long sperm storage by ant queens. We expect that PO activity will be similarly suppressed in queen spermathecae of other eusocial Hymenoptera (bees and wasps) and, more generally, of insect females that store sperm for long periods.

Sublethal Effect of Arsenic on Oxidative Stress and Antioxidant Status in Scylla serrata

Estuarine edible crab, Scylla serrata (Crustacea: Decapoda) occupy diverse habitats and their strategy of survival is correlated to multidimensional immune responses and its inhabiting in bio‐unsafe environment is under constant threat of physiological stress caused by diverse xenobiotics including arsenic. Experimental crabs, exposed to 1–3 ppm of sodium arsenite for a maximum period of 30 days in controlled laboratory condition indicated that arsenic as cytotoxicant as well as immunotoxicant causes damage and an alteration in cytotoxic status and antioxidant status in hemocytes. In this study, we investigated the intrahemocytic production of phenoloxidase, super oxide anion, nitric oxide, and its subsequent scavenging under the exposure of sodium arsenite. Arsenic induced variations in generation of cytotoxic molecules as well as antioxidant enzymes activity in intrahemocyte is indicative to possible immune‐alteration of the animal distributed in bio‐unsafe environment. Arsenic induced impairment of immune response may initiate an opportunistic growth of microbes in crab. Chronic exposure of S. serrata to arsenic may lead to decline of this economically important endemic species in Sundarbans Biosphere Reserve of West Bengal.

English

Bombyx mori is an important economic insect for silk production. However, many kinds of pathogens cause serious economic loss every year during sericulture. B. mori lack an acquired immune system but have a sophisticated innate immune system against foreign microorganisms. The innate immunity system of B. mori is composed of humoral and cellular immune reactions, including production of antimicrobial peptides (AMPs), phenoloxidase, phagocytosis, nodule formation, encapsulation and melanization. Three immunity pathways had been reported in the B. mori to control the gene expression and defending invading microorganisms: Toll pathway, Imd pathway and JAK-STAT pathway. It has been shown that gene families associated with recognition, modulation and effectors are conserved from vertebrates to invertebrates. Recent accomplishment in molecular immunology of B. mori has been reviewed in this paper. The development of zinc-finger nuclease technique and silkworm genome information is expected to accelerate silkworm immunity studies.   Key words: Bombyx mori, innate immunity, signal pathway, pattern recognition receptors.

Impact of Bt cotton on the immune system and histology of the midgut of the fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

Despite the efficiency of transgenic plants expressing Bacillus thuringiensis (Bt) toxins as insecticides against several lepidopterans, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is one species that presents low susceptibility to most Bt crops. This study investigated the effects of the Cry1Ac toxin expressed by Bt cotton in the midgut of S. frugiperda and its effects on the humoral and cellular immune responses. Three hypotheses were proposed and tested with contributing factors for the natural tolerance of S. frugiperda: (i) midgut regenerative cells are activated by the Cry1Ac toxin, and thus renew the epithelial cells damaged by the protein, (ii) Cry1Ac increased production of nitric oxide or phenoloxidase in the hemolymph, and (iii) there are qualitative and quantitative variations in the hemocyte levels of S. frugiperda. Caterpillars were reared using Bt cotton (Acala 90B) and non-Bt isolines (Acala 90), from the first to the fourth instar. The Bt cotton promoted elongation of the epithelial cells in the midgut of S. frugiperda caterpillars. Hence, evidence only supported the hypothesised increase of phenoloxidase (ii) and qualitative and quantitative differences in hemocyte levels (iii) in insects that were fed with Bt and non-Bt cotton. These parameters seem to explain the low susceptibility of S. frugiperda to Cry1Ac toxin and they are a viable set of responses for the evaluation of other xenobiotic factors.

Sexual dimorphism in immune response: Testing the hypothesis in an insect species with two male morphs

It has been proposed that given that males should invest in sexual traits at the expense of their investment in immune response, females are better immunocompetent than males. Typically, this idea has been tested in monomorphic species, but rarely has been evaluated in polymorphic male species. We used Paraphlebia zoe, a damselfly with two male morphs: the black-winged morph (Black-W) develop black spots as sexual traits and the hyaline-winged morph (Hyaline-W) resembles a female in size and wings color. We predicted that Black-W should have a lower immune response than Hyaline-W, but that the latter males should not differ from females in this respect. Nitric oxide (NO) and phenoloxidase (PO) production, as well as hemolymph protein content, were used as immune markers. Body size (wing length) was used as an indicator of the male condition. The results show that, as we predicted, females and Hyaline-W had higher values of NO than Black-W, corresponding to differences in size. However, the opposite was found in relation to PO production. Females had the highest levels of hemolymph protein content, whereas no differences were found between Black-W and Hyaline-W. These results partially support the sexual selection hypothesis and are discussed in the context of the life history of this species. Black-W, Hyaline-W, and females could express the immune markers that are prioritized by their particular condition, and probably neither of them could express all immune markers in an elevated manner, as this would result in an excessive accumulation of free radicals.

Screening of tree leaves as annual renewable green biomass for phenol oxidase production and biochemical characterization of mulberry (Morus alba) leaf phenol oxidases

Fruit tree leaf tissues were screened in a search for determination of an alternative source(s) for commercial phenol oxidase (PO) production considering the importance of utilization of green biomass for production of value-added products. Mulberry, pear, sour cherry and apricot leaves were identified as promising PO production sources, due to their comparable enzyme activities with respect to mushroom (Agaricus bisporus), a well-known PO source. Within the scope of this research, further biochemical characterization was only performed for mulberry (Morus alba) leaf tissue due to its high PO activity (ca. 19 EU g−1 tissue) and also its known non-toxic and edible nature which are important properties of an enzyme source to be used without detailed purification. In mulberry leaves, presence of three different PO activities, laccase, peroxidase and catechol oxidase of 62–64 kDa molecular weights, were identified. Since simple extraction/concentration steps without fractionation/purification was aimed as PO production process, operational parameters such as optimal temperature, pH and kinetic studies of overall PO activity were investigated using concentrated crude extract. The highest PO activity against 4-methyl catechol was observed at 45°C and pH 7. Michaelis–Menten kinetic parameters, Km and Vmax, of PO activity were determined as 6 mM 4-methyl catechol and 2.2 μmol quinone produced min−1 ml−1, respectively. PO activity of mulberry leaves increased up to late November. Consequently, mulberry leaves seem as a suitable PO source for industrial applications in which a wide range of substrate utilization is necessary.

Cathepsin L-like cysteine proteinase (DcCathL) from Delia coarctata (wheat bulb fly): Basis of insecticidal activity

A cDNA encoding a cathepsin L-like cysteine proteinase (DcCathL) was prepared from gut tissue of larvae of wheat bulb fly ( Delia coarctata: Diptera). The predicted protein is a homologue of the product of Drosophila melanogaster gene Cp-1 (CG6692), and is similar to a sub-family of cysteine proteinases found in other insects which have roles in tissue remodelling during development, and moulting. Recombinant DcCathL was produced using the yeast Pichia pastoris as expression host, and showed hydrolytic activity in vitro towards the synthetic substrate Z-Phe-Arg-AMC with a pH optimum of 4.5. DcCathL was insecticidal to lepidopteran larvae when injected into haemolymph, causing mortality that was accompanied by systemic melanisation, suggesting that DcCathL was affecting the immune-related proteolytic activation cascade leading to production of active phenoloxidase. This process is normally negatively regulated by serpins in the haemolymph. Recombinant serpins from cabbage moth ( Mamestra brassicae) did not inhibit DcCathL, and were susceptible to degradation by the enzyme in vitro in buffer and extracted haemolymph. When M. brassicae larvae were co-injected with a lethal dose of DcCathL and exogenous recombinant serpins, no mortality or systemic melanisation was observed, suggesting that the insecticidal effects of DcCathL in vivo result from degradation of endogenous serpins.

Changes in immune effort of male field crickets infested with mobile parasitoid larvae

Insect immune defenses include encapsulation and the production of lysozymes and phenoloxidase. However, the highly mobile larvae of parasitoid Ormiine flies ( Ormia ochracea) can evade initial encapsulation, and instead co-opt host immune responses to form a critical respiratory funnel connecting them to outside oxygen. Here we ask how field crickets ( Teleogryllus oceanicus) respond immunologically to O. ochracea infestation. Host encapsulation and phenoloxidase play important roles in the formation of the respiratory funnel, so we hypothesized that decreases in these immune parameters during infestation may interfere with respiratory funnel formation and increase the likelihood of larval death. Encapsulation ability decreased after infestation with O. ochracea larvae, but phenoloxidase activity increased in both infested crickets and controls, whereas lysozyme activity decreased in infested crickets but remained constant in controls. Hosts with fewer established larvae showed greater decreases in encapsulation, and phenoloxidase activity was positively associated with the degree of larval respiratory funnel melanization. Differences between phenoloxidase and lysozyme activity in infested crickets are consistent with a trade-off within the immune system of hosts, and our results demonstrate the effects of a prior immune challenge on the ability to mount a subsequent response.

The expression of a sexually selected trait correlates with different immune defense components and survival in males of the American rubyspot

Recent studies have suggested that courtship trait expression indicates immune strength. However, most studies have measured only one immune parameter, have not assessed individual differences in immune ability according to time and have not controlled for ecological differences among individuals after an immune challenge. In this work, we tested this hypothesis and controlled for these factors using males of the American rubyspot damselfly which bear a wing red spot whose size is evolutionarily maintained via male–male territorial competition. Our general hypothesis was that territorial, large-spotted males, had a better immune ability compared to nonterritorial, small-spotted males. We expected that the following variables were greater in territorial males compared to nonterritorial males: spot size, phenoloxidase (PO) and hydrolytic enzymatic (HE) activity in males challenged and nonchallenged with a nylon implant, PO and HE activity rate; PO activity after a Serratia marcescens challenge, and survival after a nylon challenge controlling for activity and feeding differences. We found that territorial males showed larger spot areas, greater PO and HE activity (independently of whether they were challenged or not), a higher rate of PO and HE activity (but only expressed at 8 h), greater PO production after the bacterial challenge, and a higher survival after the challenge. These results corroborate that males with more pronounced sexual traits have a superior immune function.

Étude biochimique et sérotypique de 40 souches de Cryptococcus neoformans isolées de patients VIH+ à Abidjan (Côte-d'Ivoire)

Objective The prevalence of the cryptococcosis is high in Côte-d'Ivoire but the distribution of the varieties and the serological types of Cryptococcus neoformans have not previously been studied. The objective of this work was to determine the distribution of the serotypes and the biochemical characters of strains isolated in Côte-d'Ivoire. Methods Forty strains of C. neoformans isolated from the cerebrospinal fluid of Ivorian HIV-seropositive patients were studied. The isolates were cultivated on Sabouraud glucose agar with chloramphenicol and on Pal's modified-medium and incubated at 37 °C for 48 hours. The biochemical characters were studied using the ID 32C gallery and the APIZYM gallery (bioMérieux ®, Marcy-l'Etoile, France), the production of phenol oxidase and urease and the reduction of nitrate. Serological typing was performed by agglutination (Crypto Check ®, Iatron Laboratories Inc., Tokyo, Japan). Results The study revealed that 100% of the strains belonged to C. neoformans var. neoformans, 95% to serotype A and 5% to serotype D. The auxanogram test revealed 6 profiles of which 3 were the most frequently represented and included 79% of the strains. Five specific substrates were not assimilated by any strain. The enzymatic profiles performed with the APIZYM gallery including 19 substrates was able to reveal the non expression of 9 enzymes and varying degrees of expression of the 10 other enzymes. Conclusion C. neoformans var. neoformans serotype A was the main agent responsible for cryptococcal meningitis in HIV-serpositive patients in Abidjan (Côte-d'Ivoire).

Relationship of Phenol Oxidase Activity and Hydroxyl-Radical Generation to Wood Degradation by White-Rot Basidiomycetes

Wood degradation, phenol oxidase activity, and the generation of hydroxyl radicals were examined in cultures of eight basidiomycetes that are usually regarded as white-rot fungi. Six of the fungi degraded Japanese beech wood to a significant extent under the conditions used. These six basidiomycetes also had measurable phenol oxidase activity after 1wk in woodcontaining cultures. With two of the six fungi, phenol oxidase activity peaked after 1wk of incubation. One of the six fungi had the highest activity after 6wk. The other three fungi had relatively constant phenol oxidase activities throughout the 8 wk incubation period. The six basidiomycetes that significantly degraded Japanese beech wood also generated significant amounts of hydroxyl radicals in the wood-containing cultures. The combined production of phenol oxidase and hydroxyl radicals in intact cultures of the eight basidiomycetes correlated roughly with the degree of wood degradation in the cultures. Most of the hydroxyl radicals were produced by agents other than phenol oxidase in cultures of white-rot fungi.

Effects of β-carotene source,Dunaliella salina, and astaxanthin on pigmentation, growth, survival and health ofPenaeus monodon

We report the results of controlled feeding trials on the tiger shrimp, Penaeus monodon (Fabricius). The desired colour was obtained by feeding a diet supplemented with astaxanthin (50 p.p.m) for 7–8 weeks before harvest. Similar coloration was achieved, however, by including in the feed an algal material (Dunaliella salina) that contains β-carotene but no astaxanthin. Feed containing β-carotene at 125 p.p.m also gave the required coloration at 7–8 weeks, whereas the same results was obtained after only 5–6 weeks when feed containing 175 p.p.m β-carotene was used. Irrespective of whether the shrimp were fed astaxanthin or β-carotene, the main carotenoid accumulation was astaxanthin, in free and esterified form, showing that P. monodon has the metabolic ability to convert β-carotene into astaxanthin. The supplementation with β-carotene or astaxanthin had no significant effect on growth, average final weight, survival rate or feed conversion ratio, nor on the immune response of the shrimp in terms of production of haemocytes phenoloxidase in the cytoplasmic granules of the haemocytes, microbial clearance ability and resistance to infectious diseases. This is a highly significant development for commercial aquaculture, because it shows that supplementing diets with β-carotene can achieve the same result as doing so with much more expensive astaxanthin.

An isolate of Rhizopus nigricans capable of tolerating and removing pentachlorophenol

Rhizopus nigricans, isolated from an industrial effluent (paper mill), was resistant to pentachlorophenol (PCP) in Petri dishes and in submerged cultures (100 and 25 mg l−1 respectively). It was shown that this strain of R. nigricans can remove PCP in submerged culture. When 12.5 mg of PCPl−1 were added at 48 h, this compound had been completely removed by 144h. Results indicated that the fungus did not produce extracellular lignin peroxidase (LiP) and laccase, but extracellular phenoloxidase production was observed. The synthesis of the latter enzyme was stimulated by the presence of PCP and/or tyrosine. These results indicate that this fungus, and probably other filamentous fungi, have an interesting potential to be used in processes for chlorophenol biodegradation.

Production and characterization of thermostable phenol oxidases of the ascomycete Thermoascus aurantiacus

The effect of some culture conditions on extracellular phenol oxidase (PO) production by the thermophilic ascomycete Thermoascus aurantiacus was studied. The effect of different lignocellulosic and an aromatic compound (p‐anisidine) as inducers of PO activity was studied in liquid culture. The most effective substrate, wheat bran, induced between 1 and 2 units/ml PO activity when utilized at a concentration of 1.5% (w/v). The type and size of fungal inoculum influenced the PO production. However, the agitation of cultures did not increase PO production. An initial pH between 6 and 8 resulted in a higher PO production. The crude extracts of T. aurantiacus had activities sufficient to oxidize various typical substrates of POs in the absence of H2O2. Higher activities were attained with 2, 2'‐azinobis‐(3‐ethylthiazoline 6‐sulphonate), 2, 6‐dimethoxyphenol and o‐dianisidine. Typical inhibitors of metal‐containing oxidases were the most potent inhibitors of T. aurantiacus PO. The optimal pH for o‐dianisidine oxidation was 2.8 in 50 mM citrate/phosphate buffer; the optimal oxidation temperature was between 70 and 80°C. All characteristics of the crude extract indicated that the PO is a typical fungal laccase, except for the high optimal temperature.