Effective mutations in a high redox potential laccase from Pleurotus ostreatus.

Since the first report on a laccase, there has been a notable development in the interest towards this class of enzymes, highlighted from the number of scientific papers and patents about them. At the same time, interest in exploiting laccases-mainly high redox potential-for various functions has been growing exponentially over the last 10 years. Despite decades of work, the molecular determinants of the redox potential are far to be fully understood. For this reason, interest in tuning laccase redox potential to provide more efficient catalysts has been growing since the last years. The work herein described takes advantage of the filamentous fungus Aspergillus niger as host for the heterologous production of the high redox potential laccase POXA1b from Pleurotus ostreatus and of one of its in vitro selected variants (1H6C). The system herein developed allowed to obtain a production level of 35,000 U/L (583.3 µkat/L) for POXA1b and 60,000 U/L (1,000 µkat/L) for 1H6C, corresponding to 13 and 20 mg/L for POXA1b and 1H6C, respectively. The characterised proteins exhibit very similar characteristics, with some exceptions regarding catalytic behaviour, stability and spectro-electrochemical properties. Remarkably, the 1H6C variant shows a higher redox potential with respect to POXA1b. Furthermore, the spectro-electrochemical results obtained for 1H6C make it tempting to claim that we spectro-electrochemically determined the redox potential of the 1H6C T2 site, which has not been studied in any detail by spectro-electrochemistry yet.

Exploitation of Trametes versicolor for bioremediation of endocrine disrupting chemicals in bioreactors.

Endocrine disrupting chemicals (EDCs) are environmental contaminants causing increasing concerns due to their toxicity, persistence and ubiquity. In the present study, degradative capabilities of Trametes versicolor, Pleurotus ostreatus and Phanerochaete chrysosporium to act on five EDCs, which represent different classes of chemicals (phenols, parabens and phthalate) and were first applied as single compounds, were assessed. T. versicolor was selected due to its efficiency against target EDCs and its potentialities were exploited against a mixture of EDCs in a cost-effective bioremediation process. A fed-batch approach as well as a starvation strategy were applied in order to reduce the need for input of 'fresh' biomass, and avoid the requirement for external nutrients. The fungus was successfully operated in two different bioreactors over one week. Semi-batch cultures were carried out by daily adding a mixture of EDCs to the bioreactors in a total of five consecutive degradation cycles. T. versicolor was able to efficiently remove all compounds during each cycle converting up to 21 mg L-1 day-1 of the tested EDCs. The maintained ability of T. versicolor to remove EDCs without any additional nutrients represents the main outcome of this study, which enables to forecast its application in a water treatment process.

Fungal laccases degradation of endocrine disrupting compounds.

Over the past decades, water pollution by trace organic compounds (ng/L) has become one of the key environmental issues in developed countries. This is the case of the emerging contaminants called endocrine disrupting compounds (EDCs). EDCs are a new class of environmental pollutants able to mimic or antagonize the effects of endogenous hormones, and are recently drawing scientific and public attention. Their widespread presence in the environment solicits the need of their removal from the contaminated sites. One promising approach to face this challenge consists in the use of enzymatic systems able to react with these molecules. Among the possible enzymes, oxidative enzymes are attracting increasing attention because of their versatility, the possibility to produce them on large scale, and to modify their properties. In this study five different EDCs were treated with four different fungal laccases, also in the presence of both synthetic and natural mediators. Mediators significantly increased the efficiency of the enzymatic treatment, promoting the degradation of substrates recalcitrant to laccase oxidation. The laccase showing the best performances was chosen to further investigate its oxidative capabilities against micropollutant mixtures. Improvement of enzyme performances in nonylphenol degradation rate was achieved through immobilization on glass beads.

Fungal laccases: versatile tools for lignocellulose transformation.

Conversion of lignocellulosic materials to useful, high value products normally requires a pre-treatment step to transform or deconstruct the recalcitrant and heterogeneous lignin fraction. The development of "green tools" for the transformation of lignocellulosic feedstocks is in high demand for a sustainable exploitation of such resources. This multi-faceted challenge is being addressed by an ever-increasing suite of ligninolytic enzymes isolated from various sources. Among these, fungal laccases are known to play an important role in lignin degradation/modification processes. The white-rot fungus Pleurotus ostreatus expresses multiple laccase genes encoding isoenzymes with different properties. The availability of established recombinant expression systems for P. ostreatus laccase isoenzymes has allowed to further enrich the panel of P. ostreatus laccases by the construction of mutated, "better performing" enzymes through molecular evolution techniques. New oxidative catalysts with improved activity and stability either at high temperature and at acidic and alkaline pH have been isolated and characterized.