Degradation of technical nonylphenol (t-NP), known as an endocrine-disrupting compound mixture, was assessed, using the mitosporic fungal strain UHH 1-6-18-4 isolated from nonylphenol-contaminated river water, and a strain of the aquatic hyphomycete Clavariopsis aquatica. GC-MS analysis could resolve 12 peaks attributable to nonyl chain-branched t-NP isomers. All were degraded, to individual extents. Analysis of degradation metabolites suggested intracellular hydroxylation of the nonyl moieties of individual t-NP isomers. Further metabolites also indicated shortening of branched nonyl chains, and 4-hydroxybenzoic acid was identified as a t-NP breakdown product in UHH 1-6-18-4. The t-NP degradation efficiency was higher in UHH 1-6-18-4 than in C. aquatica, and a lower specificity in degradation of individual t-NP constituents in UHH 1-6-18-4 than in C. aquatica was observed. Strain UHH 1-6-18-4 concomitantly produced extracellular laccase under degradation conditions. A mixture of CuSO 4 and vanillic acid considerably enhanced laccase production in both fungi. Laccase preparations derived from UHH 1-6-18-4 and C. aquatica cultures also converted t-NP. Laccase-catalysed transformation of t-NP led to the formation of products with higher molecular masses than that of the parent compound. These results emphasize a role of fungi occurring in aquatic ecosystems in degradation of water contaminants with endocrine activity, which has not previously been considered. Furthermore, the results are in support of two different mechanisms employed by fungi isolated from aquatic environments to initiate t-NP degradation: hydroxylation of individual t-NP isomers at their branched nonyl chains and further breakdown of the alkyl chains of certain isomers; and attack of t-NP by extracellular laccase, the latter leading to oxidative coupling of primary radical products to compounds with higher molecular masses. INTRODUCTIONNonylphenols have increasingly gained attention because of their potential to mimic the action of natural hormones in vertebrates (Ying et al., 2002). They result from incomplete biodegradation of nonylphenol polyethoxylates (NPEOs), which have been widely used as non-ionic surfactants in industrial processes and households (Braun et al., 2003;Ying et al., 2002). Both nonylphenols and NPEOs are discharged into the environment, mainly due to incomplete removal in wastewater treatment facilities (Ying et al., 2002). Nonylphenols are more resistant to biodegradation than their parent compounds and hence are found worldwide in wastewater treatment plant effluents and rivers in concentrations of up to the mg l 21 range (Heemken et al., 2001;Kolpin et al., 2002;Stachel et al., 2003;Ying et al., 2002). Due to their hydrophobicity, they tend to adsorb onto surface water particles and sediments and accumulate in aquatic organisms (Heemken et al., 2001;Ying et al., 2002). Consequently, nonylphenols represent a serious environmental and human health risk. The assessment of biodegradative processes affecting the environmental...

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A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer ( approximately 150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography-tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.

Abstract:The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate (CuSO 4 ). The best conditions obtained from screening experiments in shaken flasks using tomato juice, CuSO 4 , and soybean oil were integrated in an experimental design. Enhanced levels of tomato juice as the medium, CuSO 4 and soybean oil as inducers (36.8% (v/v), 3 mmol/L, and 1% (v/v), respectively) were determined for 10 L stirred tank bioreactor runs. This combination resulted in laccase titer of 143 000 IU/L (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), pH 3.0), which represents the highest activity so far reported for P. sanguineus in a 10-L fermentor. Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20 000 IU/L, whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times. Based on a partial characterization, the laccases of this strain are especially promising in terms of thermostability (half-life of 6.1 h at 60 °C) and activity titers.

Laccases are versatile biocatalysts with various potential biotechnological applications, e.g. the treatment of industrial waste waters, the detoxification of environmental pollutants, or the functionalization of renewable polymeric materials. Central composition experimental design and response surface methodology was applied to optimize the production of laccase by the aquatic ascomycetous fungi, Phoma sp. UHH 5‐1‐03 and Coniothyrium sp. Kl‐S5, in shake flasks. A complex plant‐based medium (tomato juice) and two elicitors (Remazol Brilliant Blue R [RBBR] and CuSO4) were tested in combination at three concentrations. The highest laccase activity of 6322 ± 403 U/L was achieved on day 9 for Phoma sp. Coniothyrium sp. exerted a maximum laccase activity of 3035 ± 111 U/L on day 4. Optimal conditions were 30 % tomato juice and 450 mg/L RBBR for both strains. A concentration of 250 μM CuSO4 led to highest laccase activities in cultures of Coniothyrium sp., and 50 μM CuSO4 was most effective for Phoma sp. A remarkable synergistic effect of tomato juice and RBBR on laccase production was observed for both strains. The upscaling potential of the optimal induction conditions was demonstrated in a lab‐scale fermenter which resulted in maximum activities of 11030 ± 177 U/L on day 6 for Phoma sp. and 11530 ± 161 U/L on day 9 for Coniothyrium sp. This study therefore presents a promising alternative for laccase production in ascomycetes based on a cheap complex substrate in combination with two elicitors.