The newly isolated Clostridium beijerinckii [FeFe]-hydrogenase CbA5H was characterized by Fourier transform infrared spectroscopy coupled to enzymatic activity assays. This showed for the first time that in this enzyme the oxygen-sensitive active state H can be simply and reversibly converted to the oxygen-stable inactive H state. This suggests that oxygen sensitivity is not an intrinsic feature of the catalytic center of [FeFe]-hydrogenases (H-cluster), opening new challenging perspectives on the oxygen sensitivity mechanism as well as new possibilities for exploitation in industrial applications.

production.An extensive analysis of the expression of [FeFe]-hydrogenases in the three best producer strains was achieved by RT-PCR, covering the complete set of known genes for each species.This revealed that during H2 production there are several different [FeFe]-hydrogenases simultaneously expressed, with genes belonging to the same phylogenetic and structural classification sharing similar transcriptional profiles.

Biohydrogen and biomethane production offers many advantages for environmental protection over the fossil fuels or the existing physical-chemical methods for hydrogen and methane synthesis. The aim of this study is focused on the exploitation of several samples from the composting process: (1) a mixture of waste vegetable materials ("Mix"); (2) an unmatured compost sample (ACV15); and (3) three types of green compost with different properties and soil improver quality (ACV1, ACV2 and ACV3). These samples were tested for biohydrogen and biomethane production, thus obtaining second generation biofuels and resulting in a novel possibility to manage renewable waste biomasses. The ability of these substrates as original feed during dark fermentation was assayed anaerobically in batch, in glass bottles, in order to determine the optimal operating conditions for hydrogen and/or methane production using "Mix" or ACV1, ACV2 or ACV3 green compost and a limited amount of water. Hydrogen could be produced with a fast kinetic in the range 0.02-2.45mLH2g(-1)VS, while methane was produced with a slower kinetic in the range 0.5-8mLCH4g(-1)VS. It was observed that the composition of each sample influenced significantly the gas production. It was also observed that the addition of different water amounts play a crucial role in the development of hydrogen or methane. This parameter can be used to push towards the alternative production of one or another gas. Hydrogen and methane production was detected spontaneously from these matrices, without additional sources of nutrients or any pre-treatment, suggesting that they can be used as an additional inoculum or feed into single or two-stage plants. This might allow the use of compost with low quality as soil improver for alternative and further applications.

Four specifically designed IDE mutants have been used to unveil the molecular basis of peptidase versus E1-like activity of the enzyme.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from β-amyloid (Aβ) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aβ degradation by proteasome in a dosedependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.

Background Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial consortium as an already selected source of implementation for biomass degradation and hydrogen production. The biocatalysts investigated as mainly relevant for hydrogen production were the [FeFe]-hydrogenases expressed in Clostridia, given their very high turnover rates. Results Bio-hydrogen accumulation was related to the modulation of gene expression of multiple [FeFe]-hydrogenases from two strains (Clostridium beijerinckii AM2 and Clostridium tyrobutyricum AM6) isolated from the same waste. Reverse Transcriptase quantitative PCR (RT-qPCR) was applied over a period of 288 h and the RT-qPCR results showed that C. beijerinckii AM2 prevailed over C. tyrobutyricum AM6 and a high expression modulation of the 6 different [FeFe]-hydrogenase genes of C. beijerinckii in the first 23 h was observed, sustaining cumulative hydrogen production of 0.6 to 1.2 ml H2/g VS (volatile solids). These results are promising in terms of hydrogen yields, given that no pre-treatment was applied, and suggested a complex cellular regulation, linking the performance of dark fermentation with key functional genes involved in bio-H2 production in presence of the autochthonous consortium, with different roles, time, and mode of expression of the involved hydrogenases. Conclusions An applicative outcome of the hydrogenases genes quantitative expression analysis can be foreseen in optimising (on the basis of the acquired functional data) hydrogen production from a nutrient-poor green waste and/or low added value compost, in a perspective of circular bioeconomy.