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 methodologies, 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.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application.

Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry.

Natural deep eutectic solvents as thermostabilizer for Humicola insolens cutinase.

As 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.

Enhancing the Thermostability of Engineered Laccases in Aqueous Betaine-Based Natural Deep Eutectic Solvents.

In recent years, natural deep eutectic solvents (NADESs) have gained increasing attention as promising nontoxic solvents for biotechnological applications, due to their compatibility with enzymes and ability to enhance their activity. Betaine-based NADESs at a concentration of 25 wt % in a buffered aqueous solution were used as media to inhibit thermal inactivation of POXA1b laccase and its five variants when incubated at 70 and 90 °C. All the tested laccases showed higher residual activity when incubated in NADES solutions, with a further enhancement achieved also for the most thermostable variant. Furthermore, the residual activity of laccases in the presence of NADESs showed a clear advantage over the use of NADESs' individual components. Molecular docking simulations were performed to understand the role of NADESs in the stabilization of laccases toward thermal inactivation, evaluating the interaction between each enzyme and NADESs' individual components. A correlation within the binding energies between laccases and NADES components and the stabilization of the enzymes was demonstrated. These findings establish the possibility of preincubating enzymes in NADESs as a facile and cost-effective solution to inhibit thermal inactivation of enzymes when exposed to high temperatures. This computer-aided approach can assist the tailoring of NADES composition for every enzyme of interest.

Immobilisation and stabilisation of glycosylated enzymes on boronic acid-functionalised silica nanoparticles.

We report a method of glycosylated enzymes' surface immobilisation and stabilisation. The enzyme is immobilised at the surface of silica nanoparticles through the reversible covalent binding of vicinal diols of the enzyme glycans with a surface-attached boronate derivative. A soft organosilica layer of controlled thickness is grown at the silica surface, entrapping the enzyme and thus avoiding enzyme leaching. We demonstrate that this approach results not only in high and durable activity retention but also enzyme stabilisation.

Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants.

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

Tailoring the specificity of the type C feruloyl esterase FoFaeC from Fusarium oxysporum towards methyl sinapate by rational redesign based on small molecule docking simulations.

The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme' active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.

Draft Genome Sequence of Talaromyces adpressus.

Here we present the draft genome sequence of the fungus Talaromyces adpressus A-T1C-84X (=CBS 142503). This strain was isolated from lignocellulosic biomass of Arundo donax during biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici, Naples, Italy.

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.

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.

Talaromyces borbonicus, sp. nov., a novel fungus from biodegraded Arundo donax with potential abilities in lignocellulose conversion.

A novel fungal species able to synthesize enzymes with potential synergistic actions in lignocellulose conversion was isolated from the biomass of Arundo donax during biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici (Naples, Italy). In this work, this species was subjected to morphological and phylogenetic analyses. Sequencing of its genome was performed, resulting in 28 scaffolds that were assembled into 27.05 Mb containing 9744 predicted genes, among which 396 belong to carbohydrate-active enzyme (CAZyme)-encoding genes. Here we describe and illustrate this previously unknown species, which was named Talaromyces borbonicus, by a polyphasic approach combining phenotypic, physiological, and sequence data.