Manganese-dependent Peroxidase Activity Research Articles

Ability of marine-derived fungi isolated from polluted saline environment for enzymatic hydrocarbon remediation.

Marine-derived fungi have attracted much attention due to their ability to present a new biosynthetic diversity. About 50 fungal isolates were obtained from Tunisian Mediterranean seawater and then screened for the presence of lignin-peroxidase (LiP), manganese-dependent peroxidase (MnP), and laccase (Lac) activities. The results obtained from both qualitative and quantitative assays showed that four of marine fungi isolates had a high potential to produce lignin-degrading enzymes. They were characterized taxonomically by a molecular method, based on international spacer (ITS) rDNA sequence analysis, as Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), and Phoma betae (MH667655.1) which have been reported as producers of ligninolytic enzyme in the literature.The enzymatic activities and culture conditions were optimized using a Fractional Factorial design (2 7- 4). Then, fungal strains were incubated with the addition of 1% of crude oil in 50% of seawater for 25days to evaluate their abilities to simultaneously degrade hydrocarbon compounds and to produce ligninolytic enzymes. The strain P. variabile exhibited the highest crude oil degradation rate (48.3%). Significant production of ligninolytic enzymes was recorded during the degradation process, which reached 2730 U/L for the MnP, 410 U/L for LiP, and 168.5 U/L for Lac. The FTIR and GC-MS analysis confirmed that the isolates rapidly biodegrade crude oil under ecological and economic conditions.

Construction of CRISPR-Cas9 genome editing platform for white-rot fungus Cerrena unicolor BBP6 and its effects on extracellular ligninolytic enzyme biosynthesis

White-rot fungi (WRF) produce extracellular ligninolytic enzymes such as laccase and manganese-dependent peroxidase (MnP). They have a remarkable potential in degrading a wide range of pollutants including polycyclic aromatic hydrocarbons and synthetic dyes. However, the lack of WRF genetic manipulation methods limits their further applications. Cerrena unicolor BBP6 is a wild-type WRF for laccase and MnP hyper production. This study is the first attempt to develop a genome editing platform for the genus Cerrena sp. based on CRISPR-Cas9 technology. Firstly, the pyrG gene in C. unicolor BBP6 was successfully disrupted and used as an effective screening marker. Subsequently, two genes, LacA and mnp3, which respectively encode a laccase and a MnP protein in strain BBP6, were disrupted. The laccase activity of the LacA mutant strain was reduced by 53.5% compared to the wild-type strain while the MnP activity of the mnp3 mutant strain was reduced by 64.7%. Our study demonstrated that the CRISPR-Cas9 genome-editing platform for C. unicolor BBP6 was successfully established, and it was effective on perturbation of ligninolytic enzyme biosynthesis by this fungus.

Fermenting and Lignin Degradability of a White-Rot Fungus Coriolopsis trogii Using Industrial Lignin as Substrate.

Bio-depolymerized the lignin macromolecules into low molecular lignin-derived aromatic compounds satisfies the requirement for carbon dioxide peaking and is also one of the important ways to realize lignin valorization. Coriolopsis trogii is a kind of less reported lignin-degrading white-rot fungus. The degradability of a self-isolated C. trogii TS01 on industrial lignins, including enzymatic hydrolysis lignin (EHL) and Kraft lignin (KL), was investigated in this paper. The results indicated that EHL could be used as an efficient carbon source to promote the cell growth and ligninolytic enzyme secretion of C. trogii TS01. Compared with using 2% glucose as carbon source, 1% EHL plus 1% glucose would increase the maximum cell dry weight, laccase activity, and manganese-dependent peroxidase activity of C. trogii TS01 by 24.8%, 164.1%, and 200%, respectively. However, the cell growth and ligninolytic enzyme secretion would be significantly inhibited in the case of 1% KL plus 1% glucose used as carbon source. As a result, at the 12th day of fermentation, the degradation rates of EHL and KL were 50.6% and 5.7%, respectively. The UV and FTIR analysis indicated that after been fermented by C. trogii TS01, S-unit content in EHL was decreased by 12.5% but G-unit content was increased by 53.7%. In conclusion, the research of this paper will provide a promising solution for the valorization of enzymatic hydrolysis lignin since the high biodegradation rate of lignin and high activity of ligninolytic enzymes could be achieved simultaneously.

Bioremediation of polycyclic aromatic hydrocarbons from an aged contaminated agricultural soil using degrading bacteria and soil amendments

The combined effects of degrading bacteria (Paracoccus sp. LXC), humic acid (HA), and soil amendments, including chicken manure, biochar, and wormcast, were investigated through 84 d of incubation in order to improve the remediation of polycyclic aromatic hydrocarbon (PAH) in aged contaminated agricultural soil. PAH concentrations and dissipation kinetics were analyzed, and the biomass and communities of microorganisms and soil enzyme activities were examined to clarify the possible mechanism. The highest removal efficiency of PAHs was 42.1% when treated with Paracoccus sp. LXC combined with biochar and HA. PAH dissipation was consistent with the first-order kinetics model and the half-life of PAHs decreased from 2323.3 d in the natural attenuation to 103–277 d in the amended microcosms. Soil amendments and HA promoted the development of PAH-degrading bacteria and diversity of bacterial communities in soil but not that of fungi. The removal efficiency of PAHs was positively correlated with the density of PAH-degrading bacteria and soil urease (Ure), dehydrogenase (Deh), and manganese-dependent peroxidase (Mnp) activities (r = 0.824–0.941, p < .01). The Paracoccus sp. LXC apparently removed 2–4-ring PAHs, and biochar and wormcast were effective in removing 2–4-ring and 5–6-ring PAHs, respectively. The combined bioremediation strategy using degrading bacteria with soil amendments and HA has high potential for PAH removal in aged contaminated agricultural soil and the possible mechanism was the combination of bioaugmentation with Paracoccus sp. LXC and biostimulation with HA and soil amendments.

Degradation Products of Polychlorinated Biphenyls and Their In Vitro Transformation by Ligninolytic Fungi.

Metabolites of polychlorinated biphenyls (PCBs)—hydroxylated PCBs (OH-PCBs), chlorobenzyl alcohols (CB-OHs), and chlorobenzaldehydes (CB-CHOs)—were incubated in vitro with the extracellular liquid of Pleurotus ostreatus, which contains mainly laccase and low manganese-dependent peroxidase (MnP) activity. The enzymes were able to decrease the amount of most of the tested OH-PCBs by > 80% within 1 h; the removal of more recalcitrant OH-PCBs was greatly enhanced by the addition of the laccase mediator syringaldehyde. Conversely, glutathione substantially hindered the reaction, suggesting that it acted as a laccase inhibitor. Hydroxylated dibenzofuran and chlorobenzoic acid were identified as transformation products of OH-PCBs. The extracellular enzymes also oxidized the CB-OHs to the corresponding CB-CHOs on the order of hours to days; however, the mediated and nonmediated setups exhibited only slight differences, and the participating enzymes could not be determined. When CB-CHOs were used as the substrates, only partial transformation was observed. In an additional experiment, the extracellular liquid of Irpex lacteus, which contains predominantly MnP, was able to efficiently transform CB-CHOs with the aid of glutathione; mono- and di-chloroacetophenones were detected as transformation products. These results demonstrate that extracellular enzymes of ligninolytic fungi can act on a wide range of PCB metabolites, emphasizing their potential for bioremediation.

Improvement of lignocellulosic biomass conversion by optimization of fungal ligninolytic enzyme activity and molasses stillage supplementation

Fungal treatment of lignocellulose is an ecologically and economically acceptable method. However, it needs improvement to increase the hydrolysis rate. A novel combination of multiple response optimization of ligninolytic activity of Trametes hirsuta F13 and supplementation of the lignocellulosic substrate (beechwood sawdust) with sugar beet molasses stillage was employed to improve and manage the desired type of fungal ligninolytic activity leading to a significant enhancement of biomass saccharification. Obtained optimal cultivation conditions (molasses stillage concentration, 13%; substrate moisture, 63%; and temperature, 25 °C) provided the desired combination of laccase and manganese-dependent peroxidase activity, and maintained the activities longer and at higher values (51.5 ± 3.5 U/L and 91 ± 4.24 U/L, respectively) than the conditions without molasses stillage (21.5 ± 2.12 U/L and 31.5 ± 4.9 U/L, respectively). Enzyme hydrolysis of the substrate treated for 7 days in the presence of molasses stillage released 20.54 ± 0.80 mg/mL fermentable sugars, and 63.58 ± 1.47 mg/mL from 18-days treated substrate, while sugar concentrations released from the substrates pretreated without the supplementation were 16.84 ± 0.30 mg/mL and 53.63 ± 2.16 mg/mL, respectively. The proposed new approach of the lignocellulose supplementation with sugar beet molasses stillage and manipulation and improvement of fungal ligninolytic activity proved to be a promising solution for the enhancement of lignocellulose bioconversion.

Biodegradation of Reactive Orange 16 azo dye by simultaneous action of Pleurotus ostreatus and the yeast Candida zeylanoides.

The purpose was to investigate a simultaneous biodegradation of the recalcitrant monoazo dye Reactive Orange 16 (RO16) in a mixed culture consisting of a biofilm of Pleurotus ostreatus-colonizing polyamide carrier and a suspension of the yeast Candida zeylanoides to see their biological interactions and possible synergistic action during degradation. Decolorization in the mixed culture was more effective than in the fungal monoculture, the respective decolorizations reaching 87.5% and 70% on day 11. The proliferation of yeast was reduced compared with the C. zeylanoides monoculture but enabled the yeast to participate in decolorization. The interaction of P. ostreatus with the yeast resulted in a gradual decrease of fungal manganese-dependent peroxidase (MnP) and laccase activities. Gas chromatography-mass spectrometry (GC-MS) analysis of the degradation products brought evidence that P. ostreatus split the dye molecule asymmetrically to provide 4-(ethenylsulfonyl) benzene whose concentration was much decreased in the mixed culture suggesting its increased metabolization in the presence of the yeast. In contrast, C. zeylanoides split the azo bond symmetrically producing the metabolites 4-(ethenylsulfonyl) aniline and α-hydroxybenzenepropanoic acid. Those metabolites were rapidly degraded in the mixed culture. A novel aspect is represented by the evidence of a mutual cooperative action of the fungal and yeast microorganisms in the mixed culture resulting in rapid decolorization and degradation of the dye.

Crude Enzymatic Extract fromTrametes maximaandPaecilomyces carneusMixed Culture Entrapped on Alginate for Phenanthrene Removal in Water

The aim of this study was to evaluate application of a crude enzymatic extract from the mixed culture of Trametes maxima (T. maxima) and Paecilomyces carneus (P. carneus) in removal of phenanthrene (PHE) from water. Since PHE belong to the polycyclic aromatic hydrocarbons (PAHs), it has been used as a model compound for bioremediation treatments. PAHs are highly stable molecules, and are resistant to conventional wastewater treatment. Production of crude extract from a mixed culture of T. maxima and P. carneus does not require special conditions yet the use of 20 g/L of glycerol as carbon source enhances laccase (Lcc) and manganese-dependent peroxidase (MnP) activities. Application of crude extracts becomes feasible because it does not require further purification. The crude enzymes entrapped in alginate beads in a fixed-bed packed column reached 94.3% ± 0.7% of PHE removal after 13 h of continuous treatment under nonsterile conditions, while the alginate beads without enzymes reached only 45.3% ± 23% of PHE removal. The difference between the removal of alginate beads with and without enzymes was the oxidative activity on the surface of the alginate beads, which was observed in the increase in the C═O group and in the X-ray spectroscopy analyses of the C 1s and O 1s regions. Beads without enzymes had less C═O groups. Polyoxyethylene sorbitan monooleate and dimethyl sulfoxide also influence enzymatic activity to carry out a level of PHE removal similar to a photocatalytic treatment.

Biodegradation of unvulcanized natural rubber by microorganisms isolated from soil and rubber surface: A preliminary study

ABSTRACTThe Natural rubber (NR) biodegradation by three microorganisms has been evaluated: a yeast (Rhodotorula mucilaginosa) and a bacterium (Pseudomonas sp.), isolated in a liquid culture from soil, and a filamentous fungus (Alternaria alternata), isolated on a solid culture from an NR surface, were tested. The biodegradation was conducted for four months in liquid culture, at 30°C, in agitated and stationary conditions, using a Mineral Salt Medium with NR as the only carbon source. The growth behaviour of the yeast and the bacterium was evaluated by means of optical density measurements (OD650). At the end of the incubation, the dry weight biomass of the microorganisms was measured. R. mucilaginosa showed a higher biomass production in the agitated culture, while a more efficient production was observed in static conditions for the Pseudomonas and A. alternata strains. The highest enzymatic activity of Lignin peroxidase (LiP) and Manganese-dependent peroxidase (MnP) was obtained in static conditions for A. alternata. The laccase production was probed by guaiacol oxidative polymerization on agar plates. The microorganism biodegradation capability was assessed through a combination of SEM analysis, FTIR-ATR spectroscopy, and Size Exclusion Chromatography techniques. An extended mycelium-substrate interphase and a decrease in the NR molecular weight were observed.

Lignin-Degrading Abilities of Novel Autochthonous Fungal Isolates Trametes hirsuta F13 and Stereum gausapatum F28

SUMMARYThe aim of this research is to isolate and identify fungi with high lignin-degrading abilities that are autochthonous to southern Serbian region. Two novel fungal isolates identified as Trametes hirsuta F13 and Stereum gausapatum F28 were selected to assess their ligninolytic enzyme activities and the efficiency of lignin removal from beech wood sawdust. Obtained results show that both isolates are good sources of industrially valuable enzymes with a potential for application in various biotechnological and industrial processes. Both isolates showed laccase, manganese-dependent peroxidase, and versatile peroxidase activities, while only S. gausapatum F28 had lignin peroxidase activity. This is the first record of the ability of S. gausapatum species to produce lignin peroxidase. T. hirsuta F13 showed higher laccase activity than S. gausapatum F28, while S. gausapatum F28 had higher manganese peroxidase activity. Also, T. hirsuta F13 exhibited much higher laccase activity under submerged cultivation conditions than solid-state cultivation conditions, which is rare for fungi. This is important for industrial processes since the submerged fermentation is a dominant technique in industry. The test of the efficiency of lignin removal showed that both isolates are efficient lignin decomposers. After five weeks of incubation on beech wood sawdust, the total lignin losses were 33.84% with T. hirsuta F13 and 28.8% with S. gausapatum F28.

Discoloration of the azo dye Congo Red by manganese-dependent peroxidase from Pleurotus sajor caju

This paper describes the production of ligninolytic enzymes by the white-rot fungus Pleurotus sajor caju under solid-state fermentation conditions using a cost-effective medium consisting of agro-industrial wastes. From the different agro-industrial wastes tested (i.e. orange, banana, mango and cantaloupe peels), banana peels led to the highest manganese-dependent peroxidase (MnP) activity (6.3 U/mL on the 10 day). MnP from banana peel cultures was purified and applied to the discoloration of the azo dye Congo Red (CR). The optimum temperature, pH and enzyme concentration for maximum discoloration (i.e. 95% in 1 h) were found to be 35°C, 4.0, and 1.4 U/mL, respectively. In addition, the phytotoxicity (with respect to Sorghum vulgare and Phaseolus radiatus seeds) of CR was considerably reduced after the treatment of plant material with MnP produced by P. sajor caju. The products obtained after discoloration of CR were characterized using GC/MS as 8-amino naphthol 3-sulfonic acid, 3-hydroperoxy 8-nitrosonaphthol, p-p'-dihydroxybiphenyl. Therefore, this approach holds promise for the production and application of MnP from P. sajor caju on a larger scale.

Lignocellulose degradation potential of Basidiomycota from Thrace (NE Greece)

Eighty fungal strains isolated from Northeastern Greece were evaluated for their biomass-degrading ability, both on solid and liquid cultures. Remazol Brilliant Blue R- and MnCl2-glucose plates were used for assessing ligninolytic potential. Cellulolytic and xylanolytic capabilities were evaluated with the use of Azo-CMC and Azo-Xylan, respectively, a choice that proved useful for the parallel detection of lignin degradation activities. Twenty eight strains were selected to study the production of the corresponding enzymatic activities in wheat-bran based liquid media. Most isolates produced relatively high extracellular endo-β-1,4-glucanase, endo-β-1,4-xylanase and laccase activities, whereas neither lignin nor manganese-dependent peroxidase activity was detected. The pH dependence of the measured activities was also determined. The highest cellulase and xylanase levels were detected in Trametes pubescens, while Cerrena unicolor proved to be the best laccase producer. The present work points out the great potential of Agaricomycetes (Basidiomycota) for lignocellulose degradation applications, and for many species, such as Agaricus impudicus, Coprinellus micaceus, Daedaleopsis tricolor, Fomitiporia pseudopunctata, Gymnopus foetidus, Neolentinus cyathiformis and Psathyrella fagetophila, represents the first report on the production of lignocellulolytic enzymes. Concerning fungal diversity, the species Coprinopsis spelaiophila, D. tricolor and F. pseudopunctata are reported for the first time from Greece. The strains of A. impudicus, C. spelaiophila, G. foetidus and P. fagetophila are unique as pure culture isolates.

Heterologous expression of a new manganese-dependent peroxidase gene from Peniophora incarnata KUC8836 and its ability to remove anthracene in Saccharomyces cerevisiae

The white rot fungus Peniophora incarnata KUC8836 has received an attention as the greatest degrader of polycyclic aromatic hydrocarbons (PAHs), which are hazardous xenobiotics and recalcitrant pollutants. To characterize the mechanisms through which MnP degrades PAHs, heterologous expression of manganese-dependent peroxidase (MnP) gene pimp1 was performed in Saccharomyces cerevisiae BY4741 via the pGEM-T Easy vector, resulting in the recombinant plasmid pESC-URA/pimp1 containing the MnP signal peptide. MnP was significantly secreted into the culture medium with galactose as an active protein with higher efficiency (3.58UmL-1)by transformants than by the wild-type S.cerevisiae. The recombinant MnP protein was shown to have a molecular weight of 44kDa by western blotting analysis. With regard to enhancing the bioremediation of PAHs in the environment, anthracene was effectively degraded by the MnP encoded by pimp1, with a degradation rate of 6.5% when Tween 80 was added. In addition, the MnP activity of the transformant exhibited the highest efficiency (2.49UmL-1) during the degradation. These results show that pimp1 might be useful for biodegradation and gene expression technologies at a transcriptional level, and genetic approaches can be improved by incorporating the highly ligninolytic gene pimp1 and the fungus P.incarnata KUC8836.

Lignin in Paper Mill Sludge is degraded by White-Rot Fungi in Submerged Fermentation

This study investigated biological treatment of paper mill sludge and lignin degradation by fungi. Four different Basidiomycetes white-rot fungi (WRF) were used: Coriolus versicolor, Tyromyces albidus, Trametes gallica and Pleurotus ostreatus. The fungi were cultured in submerged fermentation cultures with paper mill sludge. The pH values of the cultures and ligninolytic enzymes production profiles of the four fungi were monitored and reported here. The highest laccase activity of 202 U/L was obtained in T. albidus at day 25 while the maximum manganese-dependent peroxidase (MnP) activity of 50 U/L was obtained in C. versicolor from day 15 to 25. The P. ostreatus showed highest total cellulase activity with the peak of 0.26 μmol at day 15 while the other three WRF showed negligible total cellulase activity. The lignin contents were significantly decreased in the four WRF-treated sludge samples and were confirmed by acetyl bromide and FTIR analyses. The results suggested that lignin was preferentially used and significantly degraded or converted in all the four WRF-treated sludge samples.

Anthraquinone dyes decolorization capacity of anamorphic Bjerkandera adusta CCBAS 930 strain and its HRP-like negative mutants.

Cultures of the anamorphic fungus Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930.

Nutritional capability of and substrate suitability for Pseudogymnoascus destructans, the causal agent of bat white-nose syndrome.

Pseudogymnoascus destructans, the causal agent of bat white-nose syndrome, has caused nearly six million deaths in North American bats since its introduction into the United States in 2006. Current research has shown that caves can harbor P. destructans even after the infected bats are removed and bats no longer visit or inhabit previously infected caves. Our research focuses on elucidating reservoir requirements by investigating the nutritional capabilities of and substrate suitability requirements for six different P. destructans isolates from various localities including Illinois, Indiana, New York (Type specimen), and Pennsylvania. Enzyme assays implicate that both urease and b-glucosidase appear to be constitutive, lipase and esterase activity were more rapid than proteinase activity on 6% gelatin, gelatin degradation was accompanied by medium alkalinization, the reduction of thiosulfate generated hydrogen sulfide gas, chitinase and manganese dependent peroxidase activity were not visually demonstrated within eight weeks, and keratinase activity was not evident at pH 8 within eight weeks. We demonstrate that all P. destructans isolates are capable of growth and sporulation on dead fish, insect, and mushroom tissues. Sole nitrogen source assays demonstrated that all P. destructans isolates exhibit Class 2 nitrogen utilization and that growth-dependent interactions occur among different pH and nitrogen sources. Substrate suitability assays demonstrated that all isolates could grow and sporulate on media ranging from pH 5–11 and tolerated media supplemented with 2000 mg/L of calcium and 700 mg/L of three separated sulfur compounds: thiosulfate L-cysteine, and sulfite. All isolates were intolerant to PEG-induced matric potential with delayed germination and growth at −2.5 MPa with no visible germination at −5 MPa. Interestingly, decreasing the surface tension with Tween 80 permitted germination and growth of P. destructans in −5 MPa PEG medium within 14 days suggesting a link between substrate suitability and aqueous surface tension altering substances.

Lignocellulolytic activity of Coniophora puteana and Trametes versicolor in fermentation of wheat bran and decay of hydrothermally modified hardwoods

In submerged- (SF) and solid-state fermentation (SSF) of wheat bran (WB) substrate, the brown-rot fungus Coniophora puteana produced high levels of endoglucanase, xylanase and filter paper activity (FPA) but neither laccase nor manganese-dependent peroxidase (MnP) activity was detected. SF favored the accumulation of endoglucanase (3.2 U ml−1), xylanase (9.4 U ml−1), and FPA (0.14 U ml−1) by C. puteana. Under the same cultivation conditions, the white-rot fungus Trametes versicolor secreted lower levels of hydrolytic enzymes but expressed high laccase and MnP activities in SF (0.93 U ml−1 and 1.75 U ml−1, respectively) and SSF (1.54 U ml−1 and 1.63 U ml−1, respectively) of WB. Extracellular enzymatic activities and weight loss (WL) were investigated in untreated and hydrothermally modified (HTM) birch, aspen, and alder wood blocks, exposed to C. puteana and T. versicolor. Acetone soluble extractives and cellulose content were determined in HTM hardwoods colonized by C. puteana. WL and cellulose degradation in untreated wood correlated with the high levels of cellulase activity produced by C. puteana. HTM noticeably increased decay resistance, although it was not toxic to the fungal growth and did not disturb the secretion of the enzymes involved in wood degradation.

Involvement of extracellular and intracellular enzymes of Ceriporia sp. ZLY-2010 for biodegradation of polychlorinated biphenyls (PCBs)

This study examined the interrelation between the biodegradation of polychlorinated biphenyls (PCBs) by Ceriporia sp. ZLY-2010 and its fungal enzyme systems. The degradation rates of Aroclor 1254 and 1260 were 29.01% on day 5 and 36.80% on day 10, respectively. MnP (Manganese dependent peroxidase) and laccase activities showed the greatest increases in the samples containing Aroclors, indicating that extracellular enzymes of Ceriporia sp. ZLY-2010 were affected by the addition of Aroclors. However, the relationship between the biodegradation rate and extracellular enzymes might be obscured by the complexity of the biodegradation process. Cytochrome P450 monooxygenase was inhibited and the biodegradation rate of the Aroclor decreased by adding the inhibitor 1-aminobenzotriazole. Two-dimensional gel electrophoresis showed that intracellular enzymes play a significant role in the biodegradation of Aroclor. Complex extracellular and intracellular enzyme systems in Ceriporia sp. ZLY-2010 play an important role in degrading PCBs. Physiological changes of Ceriporia sp. ZLY-2010 caused by PCBs appeared to affect biodegradation of PCBs. However, it is necessary to further study the unidentified enzymes related to the biodegradation of Aroclor.

Mechanistic Study of 17α-Ethinylestradiol Biodegradation by Pleurotus ostreatus: Tracking of Extracelullar and Intracelullar Degradation Mechanisms

The white rot fungus Pleurotus ostreatus is able to completely remove the synthetic hormone 17α-ethinylestradiol (EE2, 200 μg in 20 mL) from a liquid complex or mineral medium in 3 or 14 days, respectively. Its efficiency has also been documented in the removal of estrogenic activity that correlated with the EE2 degradation. A set of in vitro experiments using various cellular and enzyme fractions has been performed and the results showed that EE2 was degraded by isolated laccase (about 90% within 24 h). The degradation was also tested with concentrated extracellular liquid where degradation reached 50% mainly due to the laccase activity; however, after a supplementation with H₂O₂ and Mn²⁺, residual manganese-dependent peroxidase activities (40 times lower than Lac) raised the degradation to 100%. Moreover, the intracellular fraction and also laccase-like activity associated with fungal mycelium were found to be efficient in the degradation too. Isolated microsomal proteins appeared to also be involved in the process. The degradation was completely suppressed in the presence of cytochrome P-450 inhibitors, piperonylbutoxide and carbon monoxide, indicating a role of this monooxygenase in the degradation process. Attention was also paid to monitoring of changes in the estrogenic activity during these particular in vitro experiments when mainly degradations related to ligninolytic enzymes were found to decrease the estrogenic activity with EE2 removal proportionally. Several novel metabolites of EE2 were detected using different chromatographic method with mass spectrometric techniques (LC-MS, GC-MS) including also [¹³C]-labeled substrates. The results document the involvement of various different simultaneous mechanisms in the EE2 degradation by P. ostreatus by both the ligninolytic system and the eukaryotic machinery of cytochromes P-450.

Production of lignocellulose-degrading enzymes employing Fusarium solani F-552

In this work, capability of Fusarium solani F-552 of producing lignocellulose-degrading enzymes in submerged fermentation was investigated. The enzyme cocktail includes hydrolases (cellulases, xylanases, and proteinases) as well as ligninolytic enzymes: manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac). To our knowledge, this is the first report on production of MnP, LiP, and Lac together by one F. solani strain. The enzyme productions were significantly influenced by application of either lignocellulosic material or chemical inducers into the fermentation medium. Among them, corn bran significantly enhanced especially productions of cellulases and xylanases (248 and 170 U/mL, respectively) as compared to control culture (11.7 and 29.2 U/mL, respectively). High MnP activity (9.43 U/mL, control 0.45 U/mL) was observed when (+)-catechin was applied into the medium, the yield of LiP was maximal (33.06 U/mL, control 2.69 U/mL) in gallic acid, and Lac was efficiently induced by, 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] (6.74 U/mL, not detected in control). Finally, in order to maximize the ligninolytic enzymes yields, a novel strategy of introduction of mild oxidative stress conditions caused by hydrogen peroxide into the fermentation broth was tested. Hydrogen peroxide significantly increased activities of MnP, LiP, and Lac which may indicate that these enzymes could be partially involved in stress response against H(2)O(2). The concentration of H(2)O(2) and the time of the stress application were optimized; hence, when 10 mmol/L H(2)O(2) was applied at the second and sixth day of cultivation, the MnP, LiP, and Lac yields reached 21.67, 77.42, and 12.04 U/mL, respectively.