Development of novel biocatalysts and biosystems for green chemistry

The chemical synthesis currently employed in the manufacture of cosmetics presents limitations such as unwanted side reactions and the need of strong chemical conditions. In order to overcome these drawbacks, novel enzymes have been developed to catalyse the targeted bioconversions contributing to the solution of the environmental concerns of the industrial activities moving them towards sustainable biotechnologies. This work was mainly aimed at developing improved biocatalysts based on feruloyl esterases (FAEs) and glucuronoyl esterases (GEs) for the production of compounds with antioxidant activity. Novel fungal FAEs and GEs, identified through a bioinformatics approach from the analysis of 300 fungal genomes by the “Westerdijk Fungal Biodiversity Institute”, were expressed in Pichia pastoris and characterized. FAE from Aspergillus wentii was selected as the most promising enzyme to be subjected to site-directed mutagenesis to further fine-tune the enzyme towards its application in bioconversions. A homology model of this enzyme was developed and five site-directed variants were designed, expressed in P. pastoris and characterized assessing substrate specificity, solvent and thermo tolerance. This analysis led to the development of a rational designed variant with tenfold improved hydrolytic activity and a variant with enhanced thermo and solvent tolerance. As a second approach to develop improved biocatalysts based on FAEs, directed evolution was applied to the already characterized FAEs from Fusarium oxysporum (FoFaeC) and from Myceliophthora thermophila (MtFae1a). Two complete methodologies for the construction and the automated screening of evolved variants collections were developed and applied to the generation of 30,000 mutants libraries and their screening. Randomly mutated variants of FoFaeC and MtFae1a were generated through error prone-polymerase chain reaction and expressed in Yarrowia lipolytica and Saccharomyces cerevisiae, respectively. Thanks to the development of ad hoc chromogenic substrates for high-throughput assays on solid and in liquid media, screening for higher extracellular FAE activity than the wild type enzymes led to the selection of improved enzyme variants. The best evolved variants of both MtFae1a and FoFaeC were characterized for their thermotolerance, solvent tolerance and specificity towards methylated cinnamic substrates and subjected to small molecular docking studies to assess substrate interactions. In addition, MtFae1a evolved variants were tested in transesterification reactions in detergentless microemulsions for the production of target compounds selected for their potential antioxidant activity. Thus, although the screening strategy was based on the selection of evolved variants with improved hydrolytic activity, it was possible to obtain MtFae1a variants with both hydrolytic and synthetic enhanced activities to be potentially exploited in cosmetic industry. Finally, with the aim of identifying novel FAEs with non-conserved sequences, different fungal strains isolated from lignocellulosic biomasses during biodegradation under natural conditions and belonging to the microbial collection of Department of Agriculture (University of Naples “Federico II”) were screened for the production of different enzymes having potentially synergistic actions on lignocellulose conversion. This led to the selection of a novel fungal species showing FAE activity production induced by different carbon sources. Genome and transcriptome sequencing and analysis confirmed the presence of genes related to plant cell wall degrading enzymes

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

The Synthetic Potential of Fungal Feruloyl Esterases : A Correlation with Current Classification Systems and Predicted Structural Properties

Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.Peer reviewe

Fungal glucuronoyl esterases : Genome mining based enzyme discovery and biochemical characterization

4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl d-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.4-O-Methyl-D-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl D-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.Peer reviewe

Fungal feruloyl esterases: Functional validation of genome mining based enzyme discovery including uncharacterized subfamilies

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs.Peer reviewe

Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.Peer reviewe

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

Natural deep eutectic solvents as thermostabilizer for Humicola insolens cutinase

International audienceAs a new generation of green solvents, deep eutectic solvents (DESs) are considered a promising alternative to current harsh organic solvents and find application in many chemical processing methods such as extraction and synthesis. DESs, normally formed by two or more components via various hydrogen bond interactions, offer high potential as medium for biocatalysis reactions where they can improve efficiency by enhancing substrate solubility and the activity and stability of the enzymes. In the current study, the stabilization of Humicola insolens cutinase (HiC) in natural deep eutectic solvents (NADESs) was assessed. The best hydrogen bond donor among sorbitol, xylitol, erythritol, glycerol and ethylene glycol, and the best acceptor among betaine, choline chloride, choline acetate, choline dihydrogen citrate and tetramethylammonium chloride, were selected, evaluating binding energies and molecular orientations through molecular docking simulations, and finally used to prepare NADES aqueous solutions. The effects of component ratio and NADES concentration on HiC thermostability at 90 °C were also investigated. The choline dihydrogen citrate:xylitol, in a 1:1 ratio with a 20 wt% concentration, was selected as the best combination in stabilizing HiC, increasing its half-life three-fold