Lignin peroxidase is involved in the biobleaching of manganese-less oxygen-delignified hardwood kraft pulp by white-rot fungi in the solid-fermentation system

Biobleaching of manganese-less oxygen-delignified hardwood kraft pulp (E-OKP) by the white-rot fungi Phanerochaete sordida YK-624 and P. chrysosporium was examined in the solid-state fermentation system. P. sordida YK-624 possessed a higher brightening activity than P. chrysosporium, increasing pulp brightness by 13.4 points after seven days of treatment. In these fermentation systems, lignin peroxidase (LiP) activity was detected as the principle ligninolytic enzyme, and manganese peroxidase and laccase activities were scarcely detected over the course of treatment of E-OKP by either fungus. Moreover, a linear relationship between brightness increase and cumulative LiP activity was observed under all tested culture conditions with P. sordida YK-624 and P. chrysosporium. These results indicated that LiP is involved in the brightening of E-OKP by both white-rot fungi.     

Removal of estrogenic activity of endocrine-disrupting genistein by ligninolytic enzymes from white rot fungi

Endocrine-disrupting genistein was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. Genistein decreased by 93% after 4 days of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, thus suggesting that the disappearance of genistein is related to ligninolytic enzymes produced extracellularly by white rot fungi. Therefore, genistein was treated with MnP, laccase, and the laccase-mediator system with 1-hydroxybenzotriazole (HBT) as a mediator. HPLC analysis demonstrated that genistein disappeared almost completely in the reaction mixture after 4 h of treatment with either MnP, laccase, or the laccase-HBT system. Using the yeast two-hybrid assay system, it was also confirmed that three enzymatic treatments completely removed the estrogenic activity of genistein after 4h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of genistein.     

Cloning and transcriptional analysis of the gene encoding 5-aminolevulinic acid synthase of the white-rot fungus Phanerochaete sordida YK-624

In this study, we cloned the gene encoding 5-aminolevulinic acid synthase (ALAS) from the hyper-lignin-degrading fungus Phanerochaete sordida YK-624. The deduced amino acid sequence showed highest identity (93.0%) to ALAS of P. chrysosporium. Expression of the gene encoding ALAS, which we named aas, corresponded temporally with the expression and activity of manganese peroxidase.     

Degradation of polychlorinated biphenyls by extracellular enzymes of Phanerochaete chrysosporium produced in a perforated plate bioreactor

The white rot fungus Phanerochaete chrysosporium was cultivated in a perforated plate bioreactor and the expression of activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) was measured. Peak activities of the two enzymes were reached close to day 11 and therefore the cultivation was terminated on that day. Extracellular proteins were concentrated and both peroxidases separated by isoelectric focusing. Degradation of technical PCB mixtures containing low and highly chlorinated congeners (Delor 103 and Delor 106 as equivalents of Aroclor 1242 and Aroclor 1260, respectively) was performed using intact mycelium, crude extracellular liquid and enriched MnP and LiP. A decrease in PCB concentration caused by a 44-h treatment with mycelium (74% w/w for Delor 103 and 73% for Delor 106) or crude extracellular liquid (62% for Delor 103 and 58% for Delor 106) was observed. The degradation was not substrate-specific, because no significant differences between the respective degradation rates were observed with di-, tri-, tetra-, penta-, hexa-, hepta-, and octachlorinated congeners. In contrast, MnP and LiP isolated from the above-mentioned extracellular liquid did not catalyse any degradation.     

Liquid-gel partitioning using Lipidex in the determination of polychlorinated biohenyls,napthalenes, dibenzo-p-dioxins and dibenzofurans in blood plasma

A method was developed for the transfer of fat, polychlorinated biphenyls (PCBs), naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) from blood plasma into the lipophilic gel Lipidex 5000. Subsequent elution of the gel separated about 70% of the fat from the analytes. Different adsorbents and activated charcoal were applied for further purification of the sample and separation of analytes. Identification and determination of the chlorinated compounds were made by gas chromatography with electron-capture detection (GC-ECD) or gas chromatography-mass spectrometry (GC-MS). Recoveries were studied by addition of Halowax 1014 and different congeners of PCBs, PCNs, PCDDs and PCDFs to 50 ml of plasma. The mean recoveries of the individual compounds studied were 72–99%. By using the liquid-gel partitioning technique emulsions were avoided. Concentrations of lipids in plasma obtained by the present method agreed well with the concentrations obtained using liquid-liquid partitioning with chloroform-methanol.     

Trapping of 2,7-dichlorodibenzo-p-dioxin in aqueous solution by enzymatic reaction of fungal manganese peroxidase in the presence of polyunsaturated fatty acids

In the presence of polyunsaturated fatty acids, including cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), 2,7-dichlorodibenzo-p-dioxin (DCDD) was treated with manganese peroxidase (MnP) from white rot basidiomycete Phanerochaete sordida YK-624. After incubation with MnP, DCDD could not be extracted from the reaction mixture with n-hexane and was trapped in the water layer. DCDD was released by alkalification of the water layer. DCDD was also trapped after treatment with lipoxidase, which produces hydroperoxides from unsaturated lipids. The amounts of thiobarbituric acid-reactive substances produced in the MnP reactions with three highly unsaturated fatty acids were higher than the amounts produced with three fatty acids with a lower degree of unsaturation. These results suggest that a DCDD-trapping compound may be produced by peroxidation of the polyunsaturated fatty acids.     

Sorption/Desorption of Polychlorinated Dibenzo-P-Dioxins and Polychlorinated Dibenzo Furans (PCDDs/PCDFs) in the Presence of Cyclodextrins

The goal of this study was to investigate the usefulness of cyclodextrins (CDs) for the removal of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo furans (PCDDs/PCDFs) in soil and water. Five CDs having different molecular cavities and active functional groups were selected and evaluated for their ability to include (trap) PCDDs/PCDFs in soil and water. For the soil experiments, CDs were added to the soil on day one and the concentrations of unbound PCDDs/PCDFs were monitored over a 28-day period. Parallel control experiments were conducted to assist in the process performance evaluation. The ability of CDs to remove PCDDs/PCDFs from the contaminated soil was dependent upon the type of CD used and constituents of PCDDs/PCDFs. Among the five CDs investigated, hydroxypropyl-β -cyclodextrin (HPBCD) gave the highest removal efficiency for all components of PCDDs/PCDFs. The removal efficiency of total PCDDs/PCDFs was 81% one day after application of CDs and then increased to 96% after 28 days. The α -cyclodextrin (ACD) and β -cyclodextrin (BCD) removed only 45% and 50% of the total PCDDs/PCDFs after 28 days, respectively, whereas hydroxypropyl-γ -cyclodextrin (HPGCD) removed 80% of the total PCDDs/PCDFs.     

Removal of diclofenac and mefenamic acid by the white rot fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624 and identification of their metabolites after fungal transformation

The non-steroidal anti-inflammatory drugs diclofenac (DCF) and mefenamic acid (MFA) were treated with the white rot fungus Phanerochaete sordida YK-624. DCF completely disappeared and MFA decreased by about 90% after 6 days of treatment. It was also confirmed that the fungus almost completely removed the acute lethal toxicity of DCF and MFA towards the freshwater crustacean Thamnocephalus platyurus after 6 days of treatment. Mass spectrometric and ¹H nuclear magnetic resonance analyses demonstrated that two mono-hydroxylated DCFs (4′-hydroxydiclofenac and 5-hydroxydiclofenac) and one di-hydroxylated DCF (4′,5-dihydroxydiclofenac) were formed via fungal transformation. The four metabolites of MFA were identified as 3′-hydroxymethylmefenamic acid (mono-hydroxylated MFA), 3′-hydroxymethyl-5-hydroxymefenamic acid (di-hydroxylated MFA), 3′-hydroxymethyl-6′-hydroxymefenamic acid (di-hydroxylated MFA) and 3′-carboxymefenamic acid. These results suggest that hydroxylation catalyzed by cytochrome P450 (CYP) in P. sordida YK-624 may be involved in the elimination and detoxification of DCF and MFA. This notion was further supported by the fact that smaller decreases in DCF and MFA were observed in cultures of P. sordida YK-624 incubated with 1-aminobenzotriazole, a known inhibitor of CYP.     

Dimerization of Bisphenol A by Hyper Lignin-Degrading Fungus Phanerochaete sordida YK-624 Under Ligninolytic Condition

Bisphenol A (BPA) was treated with hyper lignin-degrading fungus Phanerochaete sordida YK-624 under ligninolytic condition. After preculturing P. sordida YK-624 for 4 days, BPA (final concentration, 1 and 0.1 mM) was added to cultures. Both 1- and 0.1-mM BPA were effectively decreased within a 24-h treatment and two metabolites were detected. Two metabolites (5,5′-bis-[1-(4-hydroxy-phenyl)1-methyl-ethyl]-biphenyl-2,2′-diol and 4-(2-(4-hydroxy-phenyl) propan-2-yl)-2-(4-(2-(4-hydroxyphenyl) propan-2-yl) phenoxy)phenol) were identified by ESI–MS and NMR analysis. These results indicated that BPA was oxidized to BPA phenoxy radicals by ligninolytic enzymes and then dimerized at extracellular region.     

Degradation of polychlorinated biphenyl mixtures (Aroclors 1242, 1254, and 1260) by the white rot fungus Phanerochaete chrysosporium as evidenced by congener-specific analysis.

Evidence for substantial degradation of polychlorinated biphenyl mixtures Aroclor 1242, 1254, and 1260 by the white rot fungus Phanerochaete chrysosporium, based on congener-specific gas chromatographic analysis, is presented. Maximal degradation (percent by weight) of Aroclors 1242, 1254, and 1260 was 60.9, 30.5, and 17.6%, respectively. Most of the congeners in Aroclors 1242 and 1254 were degraded extensively both in low-N (ligninolytic) as well as high-N (nonligninolytic) defined media. Even more extensive degradation of the congeners was observed in malt extract medium. Congeners with varying numbers of ortho, meta, and para chlorines were extensively degraded, indicating relative nonspecificity for the position of chlorine substitutions on the biphenyl ring. Aroclor 1260, which has not been conclusively shown to undergo aerobic microbial degradation, was shown to undergo substantial net degradation by P. chrysosporium. Maximal degradation of Aroclor 1260 was observed in malt extract medium (18.4% on a molar basis), in which most of the individual congeners were degraded.     

Binding effect of polychlorinated compounds and environmental carcinogens on rice bran fiber

To accelerate the fecal excretion of polycyclic biphenyl (PCB), polychlorinated dibenzofurans (PCDFs), polychlorinated-p-dioxines (PCDDs) and various mutagens and carcinogens, their binding effect on rice bran fiber (RBF) was investigated for nine heterocyclic amines, six nitroarenes, 4-nitroquinoline-N-oxide, benzo[a]pyrene, furylfuramide, two kinds of flavonoid compounds and formaldehyde and ascorbic acid. PCBs, PCDFs and PCDDs suspended in nonane were incubated with RBF (10 mg/ml) at 37 degrees C and after centrifugation, unbound chemicals in the supernatant were analyzed by high-performance liquid chromatography (HPLC) and gas chromatography (GC). The binding effects on RBF were enhanced more than other dietary fibers (DFs), which were tested including corn, wheat bran, spinach, Hijiki (a kind of seaweed), sweet potatoes and burdock fibers. It was found that the binding effects were related to lignin contents. Binding of 3-amino-1(or 1,4)-dimethyl-5H-pyrido[4,3-b]indole (Trp-p-1 and Trp-p-2), food-derived carcinogens and 1-nitropyrene (1-NP), suspended in methanol, to RBF occurred within 10 min of incubation at 37 degrees C at pH 5-7, and decreased below pH 4; binding of food-derived carcinogens was pH dependent. The binding effects to RBF and pulp lignin were obtained at ratio of over 90%, while corn fiber and cellulose were at ratios of 4-30%. Polycyclic aromatic compounds were related to the number of rings, showing high binding effects to chemical structures with triple rings. Binding of 1-NP and PCB to RBF was not influenced in any aerobic and anaerobic bacterial cultures. It was also found that RBF was capable of binding even conjugates containing mutagens such as glucuronides and sulfates, as well as metabolites in urine. It was suggested, therefore, that mutagens and carcinogens were available for the fecal excretion of residual chemicals and their metabolites, and also for the fecal excretion of PCBs, PCDFs and related compound residues in patients of Yusho disease, who suffered food poisoning due to rice oil contaminated with PCB in Japan.     

Enhancement of biodegradation of 2,7-dichlorodibenzo-p-dioxin by addition of fungal culture filtrate

The hydrophilicity of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, increased when incubated with the culture filtrates of several strains of fungi. The possibility that the addition of these filtrates could enhance the biodegradation of 2,7-DCDD by the white-rot basidiomycetous fungus Phanerochaete sordida YK-624 was examined. The decrease of 2,7-DCDD after 3 weeks incubation in a YK-624 culture containing these filtrates was greater (30%) than that in the culture of YK-624 alone (15%). This is the first report describing the enhancement of dioxin decrease by the addition of a fungal filtrate.     

Biodegradation of γ-hexachlorocyclohexane (Lindane) by a non-white rot fungus conidiobolus 03-1-56 isolated from litter

Biodegradation of chlorinated pesticide γ-hexachlorocyclohexane (lindane) by a nonwhite rot fungus Conidiobolus 03-1-56 is reported for the first time. Conidiobolus 03-1-56, a phycomyceteous fungus isolated from litter, completely degraded lindane on the 5th day of incubation in the culture medium, and GC-ECD studies confirmed that lindane removal did not occur via adsorption on the fungal biomass. Degradation studies using different medium compositions showed that nitrogen/carbon limiting conditions (stress conditions) and presence of veratryl alcohol, induced the secretion of extracellular oxidative enzymes, which enhanced the rate of lindance biodegradation. Under optimum nutrient-limiting conditions, GC-ECD and GC-MS analysis showed complete absence of any degradation metabolite, indicating that lindane was completely mineralized. Assays for tannic acid utilization and lignin peroxidase showed similar enzymatic profiles between Conidiobolus 03-1-56 and standard white rot fungi Pleurotus ostreatus 1200 and Trametes versicolor 1086. Although Conidiobolus 03-1-56 showed a reduced enzyme activity compared to white rot fungi, preliminary evidence indicates that enzymes responsible for lignin degradation by white rots play a key role in lindane degradation by Conidiobolus 03-1-56.     

Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624

Aflatoxin B(1) (AFB(1) ) is a potent mycotoxin with mutagenic, carcinogenic, teratogenic, hepatotoxic, and immunosuppressive properties. In order to develop a bioremediation system for AFB(1) -contaminated foods by white-rot fungi or ligninolytic enzymes, AFB(1) was treated with manganese peroxidase (MnP) from the white-rot fungus Phanerochaete sordida YK-624. AFB(1) was eliminated by MnP. The maximum elimination (86.0%) of AFB(1) was observed after 48 h in a reaction mixture containing 5 nkat of MnP. The addition of Tween 80 enhanced AFB(1) elimination. The elimination of AFB(1) by MnP considerably reduced its mutagenic activity in an umu test, and the treatment of AFB(1) by 20 nkat MnP reduced the mutagenic activity by 69.2%. (1) H-NMR and HR-ESI-MS analysis suggested that AFB(1) is first oxidized to AFB(1) -8,9-epoxide by MnP and then hydrolyzed to AFB(1) -8,9-dihydrodiol. This is the first report that MnP can effectively remove the mutagenic activity of AFB(1) by converting it into AFB(1) -8,9-dihydrodiol.     

Fast automated extraction and clean-up of biological fluids for polychlorinated dibenzo-p-dioxins,dibenzofurans and coplanar polychlorinated biphenyls analysis

A fast automated extraction and clean-up procedure for low-level analysis of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (cPCBs) in biological fluids is presented. Online extraction of prepared fluids is carried out using disposable octadecyl bonded (C(18)) solid-phase extraction columns. Extracts are then cleaned up through disposable multi-layer silica (acidic, basic and neutral) and dispersed PX-21 carbon columns. This new methodology is compared with classical Soxhlet extraction and manual solid-phase extraction in terms of repeatability, reproducibility, accuracy and recovery rates for reference and certified materials. Robustness is evaluated on different matrices, such as cow's milk, breast milk and human serum. As a consequence of the reduced number of reusable glassware used, as well as lowering of solvent consumption, recorded blank levels are decreased in favor of limits of detection (LODs). Total analysis time and cost are further reduced using simultaneous sample preparation units and the sample throughput is increased compared to classical methods. As a result, this new approach appears to be suitable for the fast sample preparation often required for such fluids in case of emergency foodstuffs analysis or during large epidemiological studies.     

Physiological Role of Chlorinated Aryl Alcohols Biosynthesized De Novo by the White Rot Fungus Bjerkandera sp. Strain BOS55

The white rot fungus Bjerkandera sp. strain BOS55 produces veratryl, anisyl, 3-chloroanisyl, and 3,5-dichloroanisyl alcohol and the corresponding aldehydes de novo from glucose. All metabolites are produced simultaneously with the extracellular ligninolytic enzymes and have an important physiological function in the fungal ligninolytic system. Both mono- and dichlorinated anisyl alcohols are distinctly better substrates for the extracellular aryl alcohol oxidases than veratryl alcohol. The aldehydes formed are readily recycled by reduction by washed fungal mycelium, thus creating an extracellular H₂O₂ production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols either in the absence or in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly according to the growth conditions, indicating that production of veratryl alcohol and the production of the (chlorinated) anisyl metabolites are independently regulated. We conclude that the chlorinated anisyl metabolites biosynthesized by the white rot fungus Bjerkandera sp. strain BOS55 can be purposefully produced for ecologically significant processes such as lignin degradation.     

Bioaerosols and Particle Release During Composting of Contaminated Sawmill Soil

Compost windrows for bioremediation of soil were built at a wood-preserving site contaminated with chlorophenols, polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs). Sampling of airborne particles during the mixing of the compost windrows found concentrations of PCDDs and PCDFs in different particle sizes. The congener distribution of PCDDs and PCDFs in the collected air particle fractions was similar to that in the compost windrows, and the level of PCDDs and PCDFs was 1000-fold higher than the atmospheric background values reported previously. Viable particle-sizing samplers and several selective growth media were used to enumerate bacteria and fungi in the airborne particles. From the collected air samples, 40 bacteria were isolated and identified. Among the isolated bacteria, 80% were Gram-positive and spore-forming. Two of the identified airborne bacteria, Pseudomonas aeruginosa and Bacillus cereus, may cause human disease and are classified in biological agent hazard group 2. The amounts of airborne fungi, molds, and yeasts were 1000 to 2000 colony-forming units (CFUs) per m³. The number of actinomycetes was up to 6-fold, and the number of bacteria was 2- to 20-fold compared to background values. The overall level of airborne bacteria (200 to 3500 CFUs per m³) was low compared to the level of bacteria (10⁵ to 10⁸ CFUs per m³) found when composting municipal waste.     

Enzyme systems for biodegradation of polychlorinated dibenzo-p-dioxins

The angular dioxygenase, cytochrome P450, lignin peroxidase, and dehalogenase are known as dioxin-metabolizing enzymes. All of these enzymes have metal ions in their active centers, and the enzyme systems except for peroxidase have each distinct electron transport chain. Although the enzymatic properties of the angular dioxygenase, lignin peroxidase, and cytochrome P450 have been studied well, the information about dehalogenase is much less than other enzyme systems due to its instability under the aerobic conditions. However, this enzyme system appears to be quite promising from the viewpoint of practical use for bioremediation, because dehalogenases are capable of degradation of polychlorinated dibenzo-p-dioxins (PCDDs) with more than four chlorine substituents, whereas the other three enzyme systems prefer low-chlorinated PCDDs. On the other hand, protein engineering of angular dioxygenase, lignin peroxidase, and cytochrome P450 based on their tertiary structures has great potential to generate highly efficient dioxin-metabolizing enzymes. Actually, we successfully generated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-metabolizing enzyme by site-directed mutagenesis of cytochrome P450. We hope that recombinant microorganisms harboring genetically engineered dioxin-metabolizing enzymes will be used for bioremediation of soil contaminated with PCDDs and polychlorinated dibenzofurans in the near future.     

多氯联苯降解菌Burkholderia xenovorans LB400研究进展

Burkholderia xenovorans LB400是一株多氯联苯(polychlorinated biphenyls,PCBs)降解菌,可以氧化含有1?6个氯取代基的多氯联苯。近年来,由于其广泛的底物谱和优异的降解性能,菌株LB400已成为研究原核生物降解多氯联苯的生物化学和分子生物学方面的模式生物。目前关于PCBs的微生物降解研究已不再局限于对微生物资源的挖掘,而是更多地聚焦在LB400等降解菌的PCBs降解基因、降解酶的酶学特性以及酶的人工分子进化等方面。同时,LB400作为早期发现的降解菌,其对多氯联苯的降解途径、底物范围及相关机制也被广泛探讨;但是对于PCBs降解相关基因的调控研究较少。因此,本文以Burkholderia xenovorans LB400对多氯联苯降解为核心,通过综述其代谢途径、代谢相关基因和酶系以及降解应用等方面的研究进展,以期为深入探讨Burkholderia xenovorans LB400的应用以及进一步在遗传、分子和生化水平研究其他多氯联苯降解菌株提供借鉴。