Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells.

It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with L-3,4-dihydroxyphenylalanine (L-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the recombinant production of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme.

Industrial air pollution and mortality in the Taranto area, Southern Italy: A difference-in-differences approach.

BACKGROUND: A large steel plant close to the urban area of Taranto (Italy) has been operating since the sixties. Several studies conducted in the past reported an excess of mortality and morbidity from various diseases at the town level, possibly due to air pollution from the plant. However, the relationship between air pollutants emitted from the industry and adverse health outcomes has been controversial. We applied a variant of the "difference-in-differences" (DID) approach to examine the relationship between temporal changes in exposure to industrial PM10 from the plant and changes in cause-specific mortality rates at area unit level. METHODS: We examined a dynamic cohort of all subjects (321,356 individuals) resident in the Taranto area in 1998-2010 and followed them up for mortality till 2014. In this work, we included only deaths occurring on 2008-2014. We observed a total of 15,303 natural deaths in the cohort and age-specific annual death rates were computed for each area unit (11 areas in total). PM10 and NO2 concentrations measured at air quality monitoring stations and the results of a dispersion model were used to estimate annual average population weighted exposures to PM10 of industrial origin for each year, area unit and age class. Changes in exposures and in mortality were analyzed using Poisson regression. RESULTS: We estimated an increased risk in natural mortality (1.86%, 95% confidence interval [CI]: -0.06, 3.83%) per 1 µg/m3 annual change of industrial PM10, mainly driven by respiratory causes (8.74%, 95% CI: 1.50, 16.51%). The associations were statistically significant only in the elderly (65+ years). CONCLUSIONS: The DID approach is intuitively simple and reduces confounding by design. Under the multiple assumptions of this approach, the study indicates an effect of industrial PM10 on natural mortality, especially in the elderly population.

Role of the C-terminus of Pleurotus eryngii Ery4 laccase in determining enzyme structure, catalytic properties and stability.

The ERY4 laccase gene of Pleurotus eryngii is not biologically active when expressed in yeast. To explain this finding, we analysed the role of the C-terminus of Ery4 protein by producing a number of its different mutant variants. Two different categories of ERY4 mutant genes were produced and expressed in yeast: (i) mutants carrying C-terminal deletions and (ii) mutants carrying different site-specific mutations at their C-terminus. Investigation of the catalytic properties of the recombinant enzymes indicated that each novel variant acquired different affinities and catalytic activity for various substrates. Our results highlight that C-terminal processing is fundamental for Ery4 laccase enzymatic activities allowing substrate accessibility to the enzyme catalytic core. Apparently, the last 18 amino acids in the C-terminal end of the Ery4 laccase play a critical role in enzyme activity, stability and kinetic and, in particular biochemical and structural data indicate that the K532 residue is fundamental for enzyme activation. These studies shed light on the structure/function relationships of fungal laccases and will enhance the development of biotechnological strategies for the industrial exploitation of these enzymes.