From Waste to Value: Recent Insights into Producing Vanillin from Lignin.

Vanillin, one of the most widely used and appreciated flavoring agents worldwide, is the main constituent of vanilla bean extract, obtained from the seed pods of various members belonging to the Orchidaceae family. Due to the great demand in the food confectionery industry, as well as in the perfume industry, medicine, and more, the majority of vanillin used today is produced synthetically, and only less than one percent of the world's vanilla flavoring market comes directly from the traditional natural sources. The increasing global demand for vanillin requires alternative and overall sustainable new production methods, and the recovery from biobased polymers, like lignin, is an environmentally friendly alternative to chemical synthesis. The present review provides firstly an overview of the different types of vanillin, followed by a description of the main differences between natural and synthetic vanillin, their preparation, the market of interest, and the authentication issues and the related analytical techniques. Then, the review explores the real potentialities of lignin for vanillin production, presenting firstly the well-assessed classical methods and moving towards the most recent promising approaches through chemical, biotechnological and photocatalytic methodologies, together with the challenges and the principal issues associated with each technique.

Phospholipase D Immobilization on Lignin Nanoparticles for Enzymatic Transformation of Phospholipids.

Lignin nanoparticles (LNPs) are promising components for various materials, given their controllable particle size and spherical shape. However, their origin from supramolecular aggregation has limited the applicability of LNPs as recoverable templates for immobilization of enzymes. In this study, we show that stabilized LNPs are highly promising for the immobilization of phospholipase D (PLD), the enzyme involved in the biocatalytic production of high-value polar head modified phospholipids of commercial interest, phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine. Starting from hydroxymethylated lignin, LNPs were prepared and successively hydrothermally treated to obtain c-HLNPs with high resistance to organic solvents and a wide range of pH values, covering the conditions for enzymatic reactions and enzyme recovery. The immobilization of PLD on c-HLNPs (PLD-c-HLNPs) was achieved through direct adsorption. We then successfully exploited this new enzymatic preparation in the preparation of pure polar head modified phospholipids with high yields (60-90 %). Furthermore, the high stability of PLD-c-HLNPs allows recycling for a number of reactions with appreciable maintenance of its catalytic activity. Thus, PLD-c-HLNPs can be regarded as a new, chemically stable, recyclable and user-friendly biocatalyst, based on a biobased inexpensive scaffold, to be employed in sustainable chemical processes for synthesis of value-added phospholipids.

Dependence of 1H-NMR T 1 relaxation time of trimethylglycine betaine deep eutectic solvents on the molar composition and on the presence of water.

1H-NMR spin lattice relaxation times (T 1), measured by inversion recovery technique, allowed to establish the stoichiometric coefficient (ratio between the H-bond acceptor and H-bond donor) of a series of trimethylglycine betaine/diol based deep eutectic solvents (DESs); ethylene glycol, triethylene glycol and 1,3-propandiol were selected as H-bond donors. The maximum amount of water tolerated by the DES, before its complete hydration, was determined as well. Finally, the method was validated comparing the eutectic composition of the betaine/glycol system with that determined by means of differential scanning calorimetry analysis; the stoichiometric coefficients were identical.

Fractionation of Raw and Parboiled Rice Husks with Deep Eutectic Solvents and Characterization of the Extracted Lignins towards a Circular Economy Perspective.

In the present work, rice husks (RHs), which, worldwide, represent one of the most abundant agricultural wastes in terms of their quantity, have been treated and fractionated in order to allow for their complete valorization. RHs coming from the raw and parboiled rice production have been submitted at first to a hydrothermal pretreatment followed by a deep eutectic solvent fractionation, allowing for the separation of the different components by means of an environmentally friendly process. The lignins obtained from raw and parboiled RHs have been thoroughly characterized and showed similar physico-chemical characteristics, indicating that the parboiling process does not introduce obvious lignin alterations. In addition, a preliminary evaluation of the potentiality of such lignin fractions as precursors of cement water reducers has provided encouraging results. A fermentation-based optional preprocess has also been investigated. However, both raw and parboiled RHs demonstrated a poor performance as a microbiological growth substrate, even in submerged fermentation using cellulose-degrading fungi. The described methodology appears to be a promising strategy for the valorization of these important waste biomasses coming from the rice industry towards a circular economy perspective.

Tuning Lignin Characteristics by Fractionation: A Versatile Approach Based on Solvent Extraction and Membrane-Assisted Ultrafiltration.

Technical lignins, typically obtained from the biorefining of lignocellulosic raw materials, represent a highly abundant natural aromatic feedstock with high potential in a sustainable economy scenario, especially considering the huge primary production volumes and the inherently renewable nature of this resource. One of the main drawbacks in their full exploitation is their high variability and heterogeneity in terms of chemical composition and molecular weight distribution. Within this context, the availability of effective and robust fractionation processes represents a key requirement for the effective valorization of lignin. In the present work, a multistep fractionation of two different well known technical lignins obtained from two distinct delignification processes (soda vs. kraft pulping) was described. A comprehensive approach combining solvent extraction in organic or aqueous medium with membrane-assisted ultrafiltration was developed in order to maximize the process versatility. The obtained lignin fractions were thoroughly characterized in terms of their chemical, physical, thermal, and structural properties, highlighting the ability of the proposed approach to deliver consistent and reproducible fractions of well-controlled and predictable characteristics, irrespective of their biomass origin. The results of this study demonstrate the versatility and the reliability of this integrated multistep fractionation method, which can be easily adapted to different solvent media using the same ultrafiltration membrane set up, thereby enhancing the potential applicability of this approach in an industrial scale-up perspective for a large variety of starting raw lignins.

Two-Step Fractionation of a Model Technical Lignin by Combined Organic Solvent Extraction and Membrane Ultrafiltration.

A fractionation method for technical lignin was developed, combining organic solvent extraction and membrane ultrafiltration of the solvent soluble component. This method was validated on a commercial wheat straw/Sarkanda grass lignin (Protobind 1000) using 2-butanone (MEK) as the solvent for both the extraction and the ultrafiltration operations. The parent lignin and the different obtained fractions were fully characterized in terms of chemical composition and physicochemical properties by gel permeation chromatography, gas chromatography/mass spectrometry (GC/MS), pyrolysis-GC/MS, total phenol contents, 31P nuclear magnetic resonance (31P NMR), thermogravimetric analysis, differential scanning calorimetry analysis, and Fourier-transform infrared spectroscopy. The results show that the proposed process allows a straightforward recovery of the different lignin fractions as well as a selective control over their molecular mass distribution and related dependent properties. Moreover, the operating flexibility of the Soxhlet/ultrafiltration process allows the treatment of lignins from different feedstocks using the same installation just by modulating the choice of the solvent and the membrane porosity with the best characteristics. This is one of the most important features of the proposed strategy, which represents a new fractionation approach with the potential to improve lignin valorization for materials science and preparative organic chemistry applications.

A novel, simple screening method for investigating the properties of lignin oxidative activity.

Enzymatic lignin degradation represents a key challenge for integrated biorefineries. Notwithstanding the rich content in aromatic compounds, lignin's complex structure has hampered identification of an effective and cost-efficient enzymatic procedure to transform it into less complex product families. Advancements in enzymatically modifying or degrading lignin require a simple and reliable analytical method to quickly screen diverse lignin samples by employing different enzymes and conditions. Here, we report on a novel, rapid, and economic colorimetric assay for lignin oxidation based on the reaction of 2,4-dinitrophenylhydrazine with the carbonyl groups generated by enzymatic oxidation. The assay was validated on monomeric and dimeric lignin model compounds by comparison with HPLC analysis. The colorimetric method was used to investigate the activity of ten laccases and eight peroxidases on three technical lignins under different experimental conditions (e.g., by altering pH and mediator used). The colorimetric method was also coupled to a size-exclusion chromatographic separation of the lignin sample obtained after the enzymatic treatment in order to verify whether the enzymatic treatment resulted in lignin depolymerization, too. On the basis of this novel procedure, appropriate enzymatic treatments can now be identified to generate valuable lignin product streams.

Aminoacetone oxidase from Streptococcus oligofermentans belongs to a new three-domain family of bacterial flavoproteins.

The aaoSo gene from Streptococcus oligofermentans encodes a 43 kDa flavoprotein, aminoacetone oxidase (SoAAO), which was reported to possess a low catalytic activity against several different L-amino acids; accordingly, it was classified as an L-amino acid oxidase. Subsequently, SoAAO was demonstrated to oxidize aminoacetone (a pro-oxidant metabolite), with an activity ~25-fold higher than the activity displayed on L-lysine, thus lending support to the assumption of aminoacetone as the preferred substrate. In the present study, we have characterized the SoAAO structure-function relationship. SoAAO is an FAD-containing enzyme that does not possess the classical properties of the oxidase/dehydrogenase class of flavoproteins (i.e. no flavin semiquinone formation is observed during anaerobic photoreduction as well as no reaction with sulfite) and does not show a true L-amino acid oxidase activity. From a structural point of view, SoAAO belongs to a novel protein family composed of three domains: an α/ß domain corresponding to the FAD-binding domain, a ß-domain partially modulating accessibility to the coenzyme, and an additional α-domain. Analysis of the reaction products of SoAAO on aminoacetone showed 2,5-dimethylpyrazine as the main product; we propose that condensation of two aminoacetone molecules yields 3,6-dimethyl-2,5-dihydropyrazine that is subsequently oxidized to 2,5-dimethylpyrazine. The ability of SoAAO to bind two molecules of the substrate analogue O-methylglycine ligand is thought to facilitate the condensation reaction. A specialized role for SoAAO in the microbial defence mechanism related to aminoacetone catabolism through a pathway yielding dimethylpyrazine derivatives instead of methylglyoxal can be proposed.

Design of a novel LOX-1 receptor antagonist mimicking the natural substrate.

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is overexpressed in atherosclerotic lesions. LOX-1 specific inhibitors, urgently necessary to reduce the rate of atherosclerotic and inflammation processes, are not yet available. We have designed and synthesized a new modified oxidized phospholipid, named PLAzPC, which plays to small scale the ligand-receptor recognition scheme. Molecular docking simulations confirm that PLAzPC disables the hydrophobic component of the ox-LDL recognition domain and allows the interaction of the l-lysine backbone charged groups with the solvent and with the charged/polar residues located around the edges of the LOX-1 hydrophobic tunnel. Binding assays, in a cell model system expressing human LOX-1 receptors, confirm that PLAzPC markedly inhibits ox-LDL binding to LOX-1 with higher efficacy compared to previously identified inhibitors.

Multistep enzyme catalyzed reactions for unnatural amino acids.

The use of unnatural amino acids, particularly synthetic α-amino acids, for modern drug discovery research requires the availability of enantiomerically pure isomers. Starting from a racemate, one single enantiomer can be obtained using a deracemization process. The two more common strategies of deracemization are those obtained by stereoinversion and by dynamic kinetic resolution. Both techniques will be here described using as a substrate the D,L-3-(2-naphthyl)-alanine, a non-natural amino acid: the first one employing a multi-enzymatic redox system, the second one combining an hydrolytic enzyme together with a base-catalyzed substrate racemization. In both cases, the final product, L-3-(2-naphthyl)alanine, is recovered with good yield and excellent enantiomeric excess.

Overexpression of a bacterial chymotrypsin: application for L-amino acid ester hydrolysis.

In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated L-phenylalanine and L-tyrosine methyl ester hydrolysis.

Synthesis of lysophospholipids.

New synthetic methods for the preparation of biologically active phospholipids and lysophospholipids (LPLs) are very important in solving problems of membrane-chemistry and biochemistry. Traditionally considered just as second-messenger molecules regulating intracellular signalling pathways, LPLs have recently shown to be involved in many physiological and pathological processes such as inflammation, reproduction, angiogenesis, tumorogenesis, atherosclerosis and nervous system regulation. Elucidation of the mechanistic details involved in the enzymological, cell-biological and membrane-biophysical roles of LPLs relies obviously on the availability of structurally diverse compounds. A variety of chemical and enzymatic routes have been reported in the literature for the synthesis of LPLs: the enzymatic transformation of natural glycerophospholipids (GPLs) using regiospecific enzymes such as phospholipases A1 (PLA1), A2 (PLA2) phospholipase D (PLD) and different lipases, the coupling of enzymatic processes with chemical transformations, the complete chemical synthesis of LPLs starting from glycerol or derivatives. In this review, chemo-enzymatic procedures leading to 1- and 2-LPLs will be described.

New aliphatic glycerophosphoryl-containing polyurethanes: synthesis, platelet adhesion and elution cytotoxicity studies.

in this study new poly(ether)urethanes (PeUs) based on aliphatic diisocyanates were synthesized with phospholipid-like residues as chain extenders. The primary objective was to prepare new polyurethanes from diisocyanates that are less toxic than the aromatic ones widely used in medical-grade polyurethanes, in order to investigate the effect of the different aromatic or aliphatic hard segment content on the final properties of the materials. Some glycerophospho residues were simultaneously introduced to enhance the hemocompatibility of these materials. Polymers were prepared by a conventional two-step solution polymerization procedure using hexamethylene diisocyanate (HDi) and dodecametilendiisocyanate (DDi) and poly(1,4-butanediol) with molecular weight 1000 to form prepolymers, which were subsequently polymerized with 1-glycerophosphorylcholine (1-GPC) or glycerophosphorylserine (GPS) to act as chain extenders. The reference polymers bearing 1,4-butandiol (BD) were also synthesized. The polymers obtained were characterized by fourier transform infrared spectroscopy (fT-iR), nuclear magnetic resonance (1H nmR), and differential scanning calorimetry (DSC). The hemocompatibility of synthesized segmented polyurethanes was preliminarily investigated by platelet-rich plasma contact studies and related scanning electron microscopy (Sem) photographs as well as by cell viability assay after cell exposure to material elutions to assess the effect of any toxic leachables coming out from the samples. Two of the polymers gave interesting results, suggesting the desirability of further investigation into their possible use in biomedical devices.

The possible role of enantiodiscrimination in bilirubin toxicity.

The possibility that enantiodiscrimination of bilirubin might be involved in neuronal membrane destabilization, and therefore in bilirubin toxicity, was investigated, by circular dichroism, on model membranes composed of phospholipids. The equilibrium between bilirubin enantiomers is displaced at some extent by the interaction with certain phospholipids. The extent of equilibrium displacement depends on the molecular structure of phospholipids and on the state of charge of bilirubin. The results obtained validate the hypothesis of a possible involvement of chirality in bilirubin toxicity and support a previously suggested model for the molecular mechanism of the interaction of bilirubin with the synaptic membrane.

A practical selective synthesis of mixed short/long chains glycerophosphocholines.

Glycerophosphorylcholine (GPC) is transformed into the cyclic stannylene derivatives, which are selectively acylated to 1-acyl-2-lyso-glycerophosphocholines. The reaction is effective using C-2 to C-16 acid chlorides in 2-propanol. After solvent replacement the lyso-phospholipid (lyso-PL) is subjected to a second acylation using acid anhydrides in methylene chloride. A series of 1(2)-short-2(1)-long-diacyl-glycerophosphocholines are obtained in high yields and selectivity. No diacylation product was detected. In order to detect mixed-chain lipids with inverted disposition of acyl chains, the long chain was introduced first and the thus resulting isomeric compounds compared by NMR. An NMR method was developed in order to determine the positional purity of the isomeric compounds.

Tin-mediated synthesis of lyso-phospholipids.

1-O-Acyl-sn-glycero-3-phosphocholine and 1-O-acyl-sn-glycero-3-phosphoric acid have been prepared selectively and with high yields from the corresponding diols, glycerophosphoryl choline and glycerol-3-phosphate. Starting from the diols, the activated tin ketals were prepared in 2-propanol by reaction with dialkyltin oxide. The intermediates were acylated in the same solvent with long-chain fatty acid chlorides, giving the corresponding 1-acyl-lyso-phospholipids in high yield and with complete regioselectivity. The catalytic nature of the tin-mediated acylation and the relevance of the solvent are discussed.

Synthesis and antiproliferative activity of alkylphosphocholines.

Alkylphosphocholines (APC) with one or more methylene groups in the alkyl chain replaced by oxygen atoms or carbonyl groups, or both have been assembled modularly using omega-diols as central building blocks. Out of 25 new compounds of this kind, 11 were evaluated for their antiproliferative activity on four cell lines and compared with miltefosine to evaluate their hemolytic activity (HA) and cytotoxicity on non-tumoral cells (MT2), used as markers of adverse effects. Compound 13 was more active on cancer cell lines than on non-tumoral cells and the data were similar for MTT and thymidine incorporation assays. It had less HA than miltefosine. Compound 13 could therefore be a candidate for the preparation of compounds with higher cytotoxicity on cancer cells and lower general toxicity.