80 research outputs found
Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b
The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project.
V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft
In vivo and Post-synthesis Strategies to Enhance the Properties of PHB-Based Materials: A Review
The transition toward “green” alternatives to petroleum-based plastics is driven by the need for “drop-in” replacement materials able to combine characteristics of existing plastics with biodegradability and renewability features. Promising alternatives are the polyhydroxyalkanoates (PHAs), microbial biodegradable polyesters produced by a wide range of microorganisms as carbon, energy, and redox storage material, displaying properties very close to fossil-fuel-derived polyolefins. Among PHAs, polyhydroxybutyrate (PHB) is by far the most well-studied polymer. PHB is a thermoplastic polyester, with very narrow processability window, due to very low resistance to thermal degradation. Since the melting temperature of PHB is around 170–180°C, the processing temperature should be at least 180–190°C. The thermal degradation of PHB at these temperatures proceeds very quickly, causing a rapid decrease in its molecular weight. Moreover, due to its high crystallinity, PHB is stiff and brittle resulting in very poor mechanical properties with low extension at break, which limits its range of application. A further limit to the effective exploitation of these polymers is related to their production costs, which is mostly affected by the costs of the starting feedstocks. Since the first identification of PHB, researchers have faced these issues, and several strategies to improve the processability and reduce brittleness of this polymer have been developed. These approaches range from the in vivo synthesis of PHA copolymers, to the enhancement of post-synthesis PHB-based material performances, thus the addition of additives and plasticizers, acting on the crystallization process as well as on polymer glass transition temperature. In addition, reactive polymer blending with other bio-based polymers represents a versatile approach to modulate polymer properties while preserving its biodegradability. This review examines the state of the art of PHA processing, shedding light on the green and cost-effective tailored strategies aimed at modulating and optimizing polymer performances. Pioneering examples in this field will be examined, and prospects and challenges for their exploitation will be presented. Furthermore, since the establishment of a PHA-based industry passes through the designing of cost-competitive production processes, this review will inspect reported examples assessing this economic aspect, examining the most recent progresses toward process sustainability
Induction and Transcriptional Regulation of Laccases in Fungi
Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet
Neuroprotective Properties of Cardoon Leaves Extracts against Neurodevelopmental Deficits in an In Vitro Model of Rett Syndrome Depend on the Extraction Method and Harvest Time
This study investigates the bioactive properties of different extracts of cardoon leaves in
rescuing neuronal development arrest in an in vitro model of Rett syndrome (RTT). Samples were
obtained from plants harvested at different maturity stages and extracted with two different method-
ologies, namely Naviglio® and supercritical carbon dioxide (scCO2). While scCO2 extracts more
hydrophobic fractions, the Naviglio® method extracts phenolic compounds and less hydrophobic
components. Only the scCO2 cardoon leaves extract obtained from plants harvested in spring induced
a significant rescue of neuronal atrophy in RTT neurons, while the scCO2 extract from the autumn
harvest stimulated dendrite outgrowth in Wild-Type (WT) neurons. The scCO2 extracts were the
richest in squalene, 3ß-taraxerol and lupeol, with concentrations in autumn harvest doubling those in
spring harvest. The Naviglio® extract was rich in cynaropicrin and exerted a toxic effect at 20 μM
on both WT and RTT neurons. When cynaropicrin, squalene, lupeol and 3ß-taraxerol were tested
individually, no positive effect was observed, whereas a significant neurotoxicity of cynaropicrin
and lupeol was evident. In conclusion, cardoon leaves extracts with high content of hydrophobic
bioactive molecules and low cynaropicrin and lupeol concentrations have pharmacological potential
to stimulate neuronal development in RTT and WT neurons in vitro
Evidence of Unprecedented High Electronic Conductivity in Mammalian Pigment Based Eumelanin Thin Films After Thermal Annealing in Vacuum
Melanin denotes a variety of mammalian pigments, including the dark electrically conductive eumelanin and the reddish, sulfur-containing, pheomelanin. Organic (bio)electronics is showing increasing interests in eumelanin exploitation, e.g., for bio-interfaces, but the low conductivity of the material is limiting the development of eumelanin-based devices. Here, for the first time, we report an abrupt increase of the eumelanin electrical conductivity, revealing the highest value presented to date of 318 S/cm. This result, obtained via simple thermal annealing in vacuum of the material, designed on the base of the knowledge of the eumelanin chemical properties, also discloses the actual electronic nature of this material's conduction
Functionalization of Polyhydroxyalkanoates (PHA)-Based Bioplastic with Phloretin for Active Food Packaging: Characterization of Its Mechanical, Antioxidant, and Antimicrobial Activities
: The formulation of eco-friendly biodegradable packaging has received great attention during the last decades as an alternative to traditional widespread petroleum-based food packaging. With this aim, we designed and tested the properties of polyhydroxyalkanoates (PHA)-based bioplastics functionalized with phloretin as far as antioxidant, antimicrobial, and morpho-mechanic features are concerned. Mechanical and hydrophilicity features investigations revealed a mild influence of phloretin on the novel materials as a function of the concentration utilized (5, 7.5, 10, and 20 mg) with variation in FTIR e RAMAN spectra as well as in mechanical properties. Functionalization of PHA-based polymers resulted in the acquisition of the antioxidant activity (in a dose-dependent manner) tested by DPPH, TEAC, FRAR, and chelating assays, and in a decrease in the growth of food-borne pathogens (Listeria monocytogenes ATCC 13932). Finally, apple samples were packed in the functionalized PHA films for 24, 48, and 72 h, observing remarkable effects on the stabilization of apple samples. The results open the possibility to utilize phloretin as a functionalizing agent for bioplastic formulation, especially in relation to food packaging
How to enjoy laccases
An analysis of the scientific literature published in
the last 10 years reveals a constant growth of laccase applicative
research in several industrial fields followed by the
publication of a great number of patents. The Green Chemistry
journal devoted the cover of its September 2014 issue to a
laccase as greener alternative for chemical oxidation. This
indicates that laccase ‘‘never-ending story’’ has found a new
promising trend within the constant search for efficient
(bio)catalysts able tomeet the 12 green chemistry principles.A
survey of ancient and cutting-edge uses of laccase in different
industrial sectors is offered in this review with the aim both to
underline their potential and to provide inspiration for new
ones. Applications in textile and food fields have been deeply
described, as well as examples concerning polymer synthesis
and laccase-catalysed grafting. Recent applications in pharmaceutical
and cosmetic industry have also been reviewed
Microbial Processes for Upcycling Food Wastes Into Sustainable Bioplastics
Huge amounts of food waste are produced worldwide by means of agricultural processing, food shipping and storage, as well as everyday human consumption. The valorization of food wastes into a spectrum of marketable products represents a promising strategy for dealing with waste, while addressing the goals of a sustainable development, i.e. food security, environmental protection, and energy efficiency. This chapter provides insight on the latest trends in food waste valorization, focusing on their exploitation for the synthesis of microbial polyesters, the Polyhydroxyalkanoates (PHA). The recent advancements in PHA application in the field of food packaging are deepened, highlighting the main solutions adopted to customize and functionalize the synthesized materials
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