Intermediates and products of synthetic lignin (dehydrogenative polymerizate) degradation by Phlebia tremellosa.

Agitated, nitrogen-limited cultures of Phlebia tremellosa caused substantial changes in the distribution of 14C-labelled synthetic lignin (dehydrogenative polymerizate [DHP]) between water-soluble, dioxane-soluble, alkali-soluble, and insoluble fractions before much lignin carbon was metabolized to CO2. First, the insoluble form increased at the expense of the dioxane-soluble form. Later, the amounts of alkali-soluble and water-soluble 14C increased, and release of 14CO2 began. The molecular weight distribution of the dioxane-soluble lignin remained constant during degradation, but that of the water-soluble fraction changed to higher molecular weights. Culture agitation accelerated the attachment of suspended DHP to the mycelia and stimulated production of water-soluble 14C and 14CO2. The nonionic detergent Tween 80 also hastened release of 14CO2 and increased the early conversion of dioxane-soluble DHP to the alkali-soluble and insoluble forms. Oxidative polymerization is suggested as the first step in degradation of DHP by P. tremellosa.     

Decolourisation of synthetic textile dyes by Phlebia tremellosa

Phlebia tremellosa decolourised eight synthetic textile dyes (200 mg l(-1)) by greater than 96% within 14 days under stationary incubation conditions. High performance liquid chromatography analysis of culture supernatants indicated that Remazol Black B was degraded by the fungus, however, complete mineralisation did not occur as a colourless organic breakdown product accumulated. Laccase activity was detectable in culture supernatants after 5 days when the fungus was grown in the presence of an artificial textile effluent, with activity reaching a maximum of 15 U l(-1) on day 14.     

胶质射脉革菌(Phlebia tremellosa)单核菌株的制备与单核体筛选

为获得胶质射脉革菌(Phlebia tremellosa)BBEL0901的单核体,本研究首先考察了溶壁酶的浓度、菌龄、酶解时间、稳渗剂浓度等因素对原生质体产量的影响,进而采用正交试验优化影响原生质体制备的关键条件,最后基于细胞核染色的方法鉴定单核菌株,基于ITS序列对单核体进行分子鉴定后采用二代测序技术对单核体的全基...     

Mineralization and solubilization of synthetic lignin by manganese peroxidases from Nematoloma frowardii and Phlebia radiata

Crude and purified manganese peroxidase from the white-rot fungi Nematoloma frowardii and Phlebia radiata catalyzed the partial depolymerization of a [¹⁴C-ring]labelled synthetic lignin into water-soluble fragments (30–50%). The in vitro depolymerization of the ¹⁴C-labelled lignin was accompanied by a release of ¹⁴CO₂ ranging from 4 to 6%. Small quantities of the thiol mediator glutathione stimulated the depolymerization of lignin resulting in a mineralization and solubilization of up to 10 and 64%, respectively. Most of the water-soluble substances formed had molecular masses around 0.7 kDa, although a higher-molecular mass fraction was also detectable (>2 kDa). Photometric assays using 2,2′-azinobis(3-ethylbenzothiazolinesulphonate) as an indicator demonstrated that high levels of Mn(III), which were very probably responsible for the depolymerization and mineralization of the ¹⁴C-labelled lignin, were adjusted within the first 24 h of incubation. The manganese peroxidase catalyzed depolymerization process was not necessarily dependent on H₂O₂; also in the absence of the H₂O₂-generating system glucose/glucose oxidase, effective solubilization and mineralization of lignin dehydrogenation polymerizate occurred, due to the in part superoxide dismutase sensitive, ‘oxidase-like’ activity of MnP which probably produces radical species and peroxides from malonate.     

Removal of estrogenic activity from endocrine-disrupting chemicals by purified laccase of Phlebia tremellosa

A white-rot basidiomycete, Phlebia tremellosa, produced a laccase that showed increased activity during degradation of phthalates. A laccase was purified through the ion exchange chromatography and preparative gel electrophoresis, and the estimated molecular weight was 75 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 degrees C, respectively. The K(m) value of the enzyme was 55.7 microM, and the V(max) was 0.0541 OD min(-1) U(-1) for o-tolidine. Purified laccase reduced the estrogenic activity of four different endocrine-disrupting chemicals. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was involved in the removal of estrogenic activity.     

Production of lignin peroxidase and laccase by Phlebia radiata

Summary A cultivation method using carrierbound mycelium was developed for the production of lignin-modifying enzymes by Phlebia radiata. Laccase and lignin peroxidase were produced in batch and semi-continuous cultivations. Laccase activity was clearly enhanced by veratryl alcohol. The presence of both veratryl alcohol and Tween 80 was required for lignin peroxidase production in submerged cultivations. During the course of the semi-continuous cultivations production of lignin peroxidase activity increased fourfold compared with static cultivations.     

Secretion of Ligninolytic Enzymes and Mineralization of C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 muM) or elevated (24 and 120 muM) Mn(II) concentrations. However, H(2)O(2)- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of CO(2) even when a chelating agent, sodium malonate, was included in the medium.     

Secretion of Ligninolytic Enzymes and Mineralization of 14C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 μM) or elevated (24 and 120 μM) Mn(II) concentrations. However, H₂O₂- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of ¹⁴C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of ¹⁴CO₂ even when a chelating agent, sodium malonate, was included in the medium.     

Intermediates in nickel(0)-phosphine complex catalyzed dehydrogenative silylation of olefins

Nickel(0) complexes 1-4 containing π-coordinated olefin and triphenylphosphine (tricyclohexylphosphine) (starting from Ni(cod)₂) were prepared and the X-ray structures of 1 and 2 were resolved. The complexes appeared as efficient catalysts in dehydrogenative silylation of styrene and vinyltris(trimethylsiloxy)silane, but only after prior oxygenation of phosphine ligand. Stoichiometric studies of Ni(0) complexes with substrates showed that the bis(silyl)nickel(II) complex was a key intermediate in both reactions examined. A scheme of catalysis by Ni(0) complex involving olefin insertion into Ni-Si bond, as a crucial step, is presented.     

Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata

 The effects of high manganese [180 μM Mn(II)] concentration and addition of malonate (10 mM) were studied in nitrogen-limited cultures of the white-rot fungus, Phlebia radiata. High levels of manganese alone showed no systematic influence on the production of lignin peroxidase (LiP), manganese peroxidase (MnP) or laccase. In contrast, high-manganese containing cultures of P. radiata showed lower efficiency in the mineralization of ¹⁴C-ring-labelled synthetic lignin ([¹⁴C]DHP). The highest rates of mineralization, up to 30% in 18 days, were reached in low- manganese(2 μM)-containing cultures when malonate was omitted. Degradation of [¹⁴C]DHP was substantially restricted by the addition of malonate. The combination of high manganese and malonate resulted in increased levels of MnP and laccase production, whereas LiP production was repressed. Also, the profiles of expression of the MnP and LiP isozymes were affected. A new P. radiata MnP isozyme of pI 3.6 (MnP3) was found in the high-manganese cultures. Addition of malonate alone caused some repression but also stimulating effects on distinctive MnP and LiP isozymes. The results indicate that manganese and malonate are individual regulators of MnP and LiP expression and have different roles in the degradation of lignin by P. radiata. Received: 30 August 1995/Received revision: 10 January 1996/Accepted: 12 February 1996     

Biomimetic degradation of lignin and lignin model compounds by synthetic anionic and cationic water soluble manganese and iron porphyrins.

The biomimetic oxidation of 5-5' condensed and diphenylmethane lignin model compounds with several water soluble anionic and cationic iron and manganese porphyrins in the presence of hydrogen peroxide is reported. The oxidative efficiency of manganese and iron meso-tetra(2,6-dichloro-3-sulphonatophenyl) porphyrin chloride (TDCSPPMnCl and TDCSPPFeCl, respectively), meso-tetra-3-sulphonatophenyl porphyrin chloride (TSPPMnCl) and manganese meso-tetra(N-methylpyridinio)porphyrin pentaacetate (TPyMePMn(CH3COO)5) was compared on the basis of the oxidation extent of the models tested. Manganese porphyrins were found more effective in degrading lignin substructures than iron ones. Among them the cationic TPyMePMn (CH3COO)5, never used before in lignin oxidation, showed to be the best catalyst. The catalytic activity of porphyrins in hydrogen peroxide oxidation of residual kraft lignin was also investigated. The use of quantitative 31P NMR allowed the focusing on the occurrence of different degradative pathways depending on the catalyst used. TPyMePMn(CH3COO)5 was able to perform the most extensive degradation of the lignin structure, as demonstrated by the decrease of aliphatic hydroxyl groups and carboxylic acids. Noteworthy, no significant condensation reactions occurred during manganese porphyrins catalyzed oxidations of residual kraft lignin, while in the presence of iron porphyrins a substantial increase of condensed substructures was detected.     

Fungal degradation of kraft lignin and lignin sulfonates prepared form synthetic 14C-lignins

Kraft lignins (KL), bleached kraft lignins (BKL), and lignin sulfonates (LS) were prepared from synthetic ¹⁴C-lignins labeled in the aromatic nuclei or in the propyl side chains. These and control lignins (CL) were incubated with the lignin-decomposing white-rot fungus, Phanerochaete chrysosporium Burds., in a defined culture medium containing cellulose as growth substrate. Decomposition was monitored by measuring the ¹⁴CO₂ evolved. Average percentages of the [ring-¹⁴C]- and [side chain-¹⁴C]-lignins, respectively, recovered as ¹⁴CO₂ at the cessation of ¹⁴CO₂ evolution were: KL, 41 and 31; BKL, 42 and 26; LS, 28 and 21; and CL, 26 and 24. Gel permeation chromatography of radiolabeled materials extracted from spent cultures showed that substantial degradation to nonvolatile products had occurred. The polymeric components in the extracts were further degraded in fresh cultures. These results indicate that industrial lignins are significantly bioalterable, and that under favorable conditions industrial lignins are substantially biodegradable.     

Limited bacterial mineralization of fungal degradation intermediates from synthetic lignin.

The ability of selected bacterial strains and consortia to mineralize degradation intermediates produced by Phanerochaete chrysosporium from 14C-labeled synthetic lignins was studied. Three different molecular weight fractions of the intermediates were subjected to the action of the bacteria, which had been grown on a lignin-related dimeric compound. Two consortia isolated from wood being decayed naturally by a Ganoderma species of white rot fungus (the palo podrido system) mineralized 10 to 11% of the fraction with a molecular weight of approximately 500 but less than 4% of the higher- and lower-molecular-weight fractions. The consortia mineralized 5 to 9% of the original lignins. The ability of two pseudomonads isolated earlier from lignin-rich environments to mineralize the original lignins or fungus degradation products was much lower.     

Limited bacterial mineralization of fungal degradation intermediates from synthetic lignin.

The ability of selected bacterial strains and consortia to mineralize degradation intermediates produced by Phanerochaete chrysosporium from 14C-labeled synthetic lignins was studied. Three different molecular weight fractions of the intermediates were subjected to the action of the bacteria, which had been grown on a lignin-related dimeric compound. Two consortia isolated from wood being decayed naturally by a Ganoderma species of white rot fungus (the palo podrido system) mineralized 10 to 11% of the fraction with a molecular weight of approximately 500 but less than 4% of the higher- and lower-molecular-weight fractions. The consortia mineralized 5 to 9% of the original lignins. The ability of two pseudomonads isolated earlier from lignin-rich environments to mineralize the original lignins or fungus degradation products was much lower.     

Cleavages of aromatic ring and beta-O-4 bond of synthetic lignin (DHP) by lignin peroxidase

Lignin peroxidase from a white-rot basidiomycete, Phanerochaete chrysosporium, catalyzed cleavages of the aromatic ring and the beta-O-4 bond of a synthetic lignin, a dehydrogenation copolymer (DHP) of coniferyl alcohol and a (beta-O-4)-(beta-beta) lignin substructure model trimer.     

The influence of lignin degradation products on xylose fermentation by Klebsiella pneumoniae

Summary The inhibitory effects of seven closely related lignin degradation products on xylose fermentation by Klebsiella pneumoniae were studied. Compounds were added in varying concentrations. Less heavily substituted phenolics (at concentrations of, 0.1–0.4 g/l) were more inhibitory to growth and solvent production than vanillyl or syringyl derivatives. All of the cultures recovered from this inhibition after a prolonged incubation period. When the mechanism of the organism's recovery was investigated, GC and LC analysis showed that 43.5% of the vanillin was metabolized to vanillyl alcohol. Several unidentifiable compounds were also detected in trace amounts. K. pneumoniae also metabolized vanilly alcohol (54% of original supplement) and syringaldehyde; however, unlike vanillin, there was no predominant metabolite derived from these compounds. None of the metabolites derived from vanillyl alcohol could be identified while only the corresponding alcohol and trimethoxybenzene were identified among the syringaldehyde derived metabolites.     

Mutualistic degradation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether by bacteria.

Complete degradation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether is accomplished mutualistically by a two-membered bacterial culture. Bacterial isolate E1, which has been tentatively identified as an Acinetobacter, grows on veratrylglycerol-beta-(o-methoxyphenyl) ether producing guaiacol (o-methoxyphenol) as a non-metabolizable, bacteriocidal by-product. When Nocardia corallina (strain A81) is also present in media containing veratrylglycerol-beta-(o-methoxyphenyl) ether as the only carbon/energy source, it is able to grow on the guaiacol produced from veratrylglycerol-beta-(o-methoxyphenyl) ether by isolate E1. Strain A81 alone does not grow on veratrylglycerol-beta-(o-methoxyphenyl) ether. In the absence of strain A81, isolate E1 is rapidly killed by accumulated guaiacol. In the presence of the Nocardia, isolate E1 maintains its viability.     

Decay of the water reed Phragmites communis caused by the white-rot fungus Phlebia tremellosa and the influence of some environmental factors

Applied Microbiology and Biotechnology - The strain Phlebia tremellosa SBUG 1630 isolated from a thatched roof in Northern Germany is capable of colonizing and degrading effectively the water reed...     

Degradation of endocrine disrupting chemicals by genetic transformants in Irpex lacteus with an inducible laccase gene of Phlebia tremellosa

Irpex lacteus was genetically transformed using an laccase expression vector to get increased laccase producing strains. Stable integration of the vector was confirmed by PCR using the vector-specific primers, and the transformants showed increased laccase activities. When the transformants were grown with several endocrine disrupting chemicals, laccase activity of each transformant was induced up to six times higher than that of the wild type. They showed increased degrading activities against EDCs as well as increased removal rates of estrogenic activities generated by the EDCs than the wild type, and the laccase expression was increased during the degradations of the EDCs.