Carlo Morelli - Academia.edu (original) (raw)

Papers by Carlo Morelli

Food Chemistry, 2019

The industrial transformation of tomato (Lycopersicon esculentum Mill.) produces processed foods,... more The industrial transformation of tomato (Lycopersicon esculentum Mill.) produces processed foods, such as dried tomatoes. In this study two varieties (SaAb and PerBruzzo), grown in three cropping systems (one conventional and two organic ones), were processed by two types of small-scale drying (oven or sun drying), over two years of production. The dried samples were analyzed for their non-volatile and volatile composition, relating the results with sensory analysis. The multivariate analysis performed on collected data allowed a detailed comparison of the effects of processing, year-to year variation and cropping systems. Results indicated that drying methods mainly influenced the composition and flavor profile, also affected by the production year. The cropping system significantly influenced some quality indices, such as the acid and sugar amounts, and the aldehydes, respectively higher and lower in organic samples. The comprehensive PCA analysis allowed discrimination of drying methods and, to a lesser extent, cropping systems.

Natural Product Communications, 2018

Tomato is one of the most widely consumed fresh vegetables in the industrialized world and an imp... more Tomato is one of the most widely consumed fresh vegetables in the industrialized world and an important source of healthy constituents of the human diet. Despite the unique flavor characteristics of tomatoes, which make them extremely valuable in cooking, and their recognized beneficial role in the diet, the quality of tomato was traditionally only considered in connection to external appearances. As it happened with other highly requested crops, breeding programs of tomato focused their efforts on developing new varieties with higher yields and stress resistance, with better uniformity in fruit size, brighter color and prolonged shelf life. The downside of these strategies was that organoleptic features and nutritional value were often neglected, with a detrimental effect on commercial tomatoes. Over the last years, there has been an increase in consumers’ demand for tasty and healthy products. This aspect, paired with novel and multidisciplinary approaches to tomato research, allo...

European Journal of Organic Chemistry, Nov 14, 2022

γ‐Glutamyltransferases (GGTs) from different sources have been proposed in recent times as biocat... more γ‐Glutamyltransferases (GGTs) from different sources have been proposed in recent times as biocatalysts for the enzymatic synthesis of naturally occurring γ‐glutamyl derivatives with flavor‐enhancer properties and interesting biological activities. Although the enzymatic approach is considered as a viable alternative to both the troublesome and low‐yielding extraction from natural sources and synthesis through peptide chemistry requiring protection/deprotection steps, yields are not completely satisfactory, due to the intervention of GGT‐catalysed hydrolysis and autotranspeptidation side‐reactions. Here, the design and the use as biocatalyst for preparative purposes of two mutants of E. coli GGT are described. The design of mutants was pursued by docking‐guided identification of residues putatively involved in interaction with the acceptor substrate, thus probably representing a first identification of residues constituting the still elusive and poorly characterized acceptor substrate binding site of the enzyme.

\u3b3-Glutamyl dipeptides are compounds characterized by an amide bond involving the amino group ... more \u3b3-Glutamyl dipeptides are compounds characterized by an amide bond involving the amino group of one amino acid and the \u3b3-carboxyl group of a glutamic acid residues. They show interesting properties with respect to their parent amino acids. For example, the bitterness of aromatic and branched-chain amino acids used in oral dietary supplements is alleviated or even abolished upon \u3b3-glutamylation, as does the unpleasant smell of seleno amino acids, the source of the micronutrient selenium. \u3b3-Glutamyl derivatives of S-substituted cysteines are naturally occurring flavor enhancers found in garlic and onion.1 Although their possible applications render the \u3b3-glutamyl derivatives economically interesting compounds, their supply remains a problem. Isolation from natural sources, if any, is laborious and low-yielding, as their content may vary with cultivation and storage.2 Chemical synthesis is not economical, due to the need of protection/deprotection steps. A viable alternative could then rely on an enzymatic approach taking advantage by the use of a \u3b3-glutamyltransferase. \u3b3-Glutamyltransferases (GGTs, EC 2.3.2.2) are widespread, conserved enzymes found in bacteria, plants and animals.3 They catalyze the transfer of a \u3b3-glutamyl moiety from a donor compound, usually glutathione, to an acceptor amino acid through a \u3b3-glutamyl-enzyme intermediate involving a catalytically active threonine residue. As a first approach, a commercially available, crude \u3b3-glutamyltransferase of animal origin was used in our laboratories for the synthesis of some naturally occurring derivatives found in garlic.4 Then, our research group turned attention to bacterial enzymes, especially from GRAS (Generally Referred as Safe) microorganisms. The GGT from B. subtilis seemed to be well suited for our purposes and a detailed study of this enzyme was since then undertaken.5 Recent findings obtained about the enzymatic activity of B. subtilis GGT and related to the peculiar architecture of its active site will be presented, in relation to its application as a biocatalyst for the synthesis of \u3b3-glutamyl derivatives of economical interest. This work is supported by Fondazione Cariplo (TailGluTran Project, 2016-0741

MTT assays showed an intense antiproliferative activity (80%) of Aloe arborescens leaf skin extra... more MTT assays showed an intense antiproliferative activity (80%) of Aloe arborescens leaf skin extracts tested on murine myeloma cells. Bioassay-guided fractionation carried out by TLC allowed the identification of a spot showing antiproliferative activity; HPLC and NMR investigations showed that the TLC spot consisted of aloenin A and aloins A and B. The effects of the leaf extract and of the TLC spot were evaluated both by immunofluorescence techniques in order to test the microtubular array and at the morphological level by SEM and TEM observations. _____________________________________________________________________________________________________________

ChemCatChem, 2019

γ-Glutamyl transpeptidase from equine kidney (ekGGT, E.C. 2.3.2.2) is an intrinsic membrane enzym... more γ-Glutamyl transpeptidase from equine kidney (ekGGT, E.C. 2.3.2.2) is an intrinsic membrane enzyme which transfers the γglutamyl moiety of glutathione to amino acids and peptides, thus producing γ-glutamyl derivatives. An immobilization study of ekGGT was carried out with the aim to develop a robust biocatalyst for the synthesis of γ-glutamyl amino acids which are known as kokumi compounds. Heterofunctional octylglyoxyl-agarose resulted in a high immobilization yield and activity recovery (93 % and 88 %, respectively). Immobilized ekGGT retained more than 95 % activity under reaction conditions (Tris-HCl, pH 9, 0.05 M) after 6 days, whereas the residual activity after 6 reaction cycles (18 days) was 85 %. The synthesis of γ-glutamylmethionine catalyzed by octyl-glyoxylagarose-ekGGT afforded the product in 42 % yield (101 mg). The immobilized ekGGT was characterized by Raman spectroscopy. The immobilization protocol developed for ekGGT could be of general applicability to membrane proteins.

Next Generation Biocatalysis : An International Young Investigator Virtual Symposium, Feb 1, 2021

ChemSusChem

l‐Theanine (l‐Th) was synthesized by simply mixing the reactants (l‐glutamine and ethylamine in w... more l‐Theanine (l‐Th) was synthesized by simply mixing the reactants (l‐glutamine and ethylamine in water) at 25 °C and Bacillus subtilis γ‐glutamyl transferase (BsGGT) covalently immobilized on glyoxyl‐agarose according to a methodology previously reported by our research group; neither buffers, nor other additives were needed. Ratio of l‐glutamine (donor) to ethylamine (acceptor), pH, enzymatic units (IU), and reaction time were optimized (molar ratio of donor/acceptor=1 : 8, pH 11.6, 1 IU mL−1, 6 h), furnishing l‐Th in 93 % isolated yield (485 mg, 32.3 g L−1) and high purity (99 %), after a simple filtration of the immobilized biocatalyst, distillation of the volatiles (unreacted ethylamine) and direct lyophilization. Immobilized BsGGT was re‐used (four reaction cycles) with 100 % activity retention. This enzymatic synthesis represents a straightforward, fast, high‐yielding, and easily scalable approach to l‐Th preparation, besides having a favorable green chemistry metrics.

Foods

A soy protein isolate was hydrolyzed with Alcalase®, Flavourzyme® and their combination, and the ... more A soy protein isolate was hydrolyzed with Alcalase®, Flavourzyme® and their combination, and the resulting hydrolysates (A, F and A + F) were ultrafiltered and analyzed through SDS-PAGE. Fractions with MW < 1 kDa were investigated for their ACE-inhibitory activity, and the most active one (A < 1 kDa) was purified by semi-preparative RP-HPLC, affording three further subfractions. NMR analysis and Edman degradation of the most active subfraction (A1) enabled the identification of four putative sequences (ALKPDNR, VVPD, NDRP and NDTP), which were prepared by solid-phase synthesis. The comparison of their ACE-inhibitory activities suggested that the novel peptide NDRP might be the main agent responsible for A1 fraction ACE inhibition (ACE inhibition = 87.75 ± 0.61%; IC50 = 148.28 ± 9.83 μg mL−1). NDRP acts as a non-competitive inhibitor and is stable towards gastrointestinal simulated digestion. The Multiple Reaction Monitoring (MRM) analysis confirmed the presence of NDRP in A &l...

The valorization of rice bran, a by-product derived from the rice productive chain, has attracted... more The valorization of rice bran, a by-product derived from the rice productive chain, has attracted the attention of the scientific community for the production of high added-value products, because of its great availability (around 700 million tons are produced per year). The composition of rice bran is 15-22% lipids, 34-52% carbohydrates, 7-11% fibers, 8-12% moisture and 10-16% highly nutritional proteins 1 . The fatty fraction of rice bran is rich in bioactive phytochemicals that have antioxidant and chemopreventive properties 2 . Also, the protein hydrolyzates of rice bran could be used as flavor enhancers; moreover, due to their highly nutritional value and according to some studies they have a therapeutic potential 2,3 . It is a challenge though to hydrolyze the rice bran and achieve the selective separation of its useful components. In this work, a combined process for the transformation of rice bran through enzymatic catalysis is studied.

The identification of new strategies to fight bacterial infections in view of the spread of multi... more The identification of new strategies to fight bacterial infections in view of the spread of multiple resistance to antibiotics has become mandatory. It has been demonstrated that several bacteria develop poly-c-glutamic acid (c-PGA) capsules as a protection from external insults and/or host defence systems. Among the pathogens that shield themselves in these capsules are Bacillus anthracis, Francisella tularensis and several Staphylococcus strains. These are important pathogens with a profound influence on human health. The recently characterised c-PGA hydrolases, which can dismantle the c-PGA-capsules, are an attractive new direction that can offer real hope for the development of alternatives to antibiotics, particularly in cases of multidrug resistant bacteria. We have characterised in detail the cleaving mechanism and stereospecificity of the enzyme PghL (previously named YndL) from Bacillus subtilis encoded by a gene of phagic origin and dramatically efficient in degrading the long polymeric chains of c-PGA. We used X-ray crystallography to solve the three-dimensional structures of the enzyme in its zinc-free, zinc-bound and complexed forms. The protein crystallised with a c-PGA hexapeptide substrate and thus reveals details of the interaction which could explain the stereospecificity observed and give hints on the catalytic mechanism of this class of hydrolytic enzymes.

The FEBS Journal, 2018

If citing, it is advised that you check and use the publisher's definitive version for pagination... more If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections.

RSC Adv., 2016

Derivatization of γ-PGA as TBA salt assists its chemical manipulation into poly(α-alkyl-γ-glutama... more Derivatization of γ-PGA as TBA salt assists its chemical manipulation into poly(α-alkyl-γ-glutamate)s.

Natural Product Research, 2014

a-Mangostin is the major prenylated xanthone from Garcinia mangostana and it has been used also i... more a-Mangostin is the major prenylated xanthone from Garcinia mangostana and it has been used also in recent times as starting material for the semisynthetic preparation of various biologically active derivatives. Its structure is characterised by the presence of few functional groups amenable to chemical manipulations, but present in the molecule in multiple instances (three phenolic hydroxyl groups, two prenyl chains and two unsubstituted aromatic carbons). This study represents a first approach to the systematic investigation of the reactivity of a-mangostin and describes the semisynthesis of some minor xanthones isolated from G. mangostana.

Journal of Biological Chemistry, 2002

2-Phenoxyethanol is converted into phenol and acetate by a strictly anaerobic Gram-positive bacte... more 2-Phenoxyethanol is converted into phenol and acetate by a strictly anaerobic Gram-positive bacterium, Acetobacterium strain LuPhet1. Acetate results from oxidation of acetaldehyde that is the early product of the biodegradation process (Frings, J., and Schink, B. (1994) Arch. Microbiol. 162, 199-204). Feeding experiments with resting cell suspensions and 2-phenoxyethanol bearing two deuterium atoms at either carbon of the glycolic moiety as substrate demonstrated that the carbonyl group of the acetate derives from the alcoholic function and the methyl group derives from the adjacent carbon. A concomitant migration of a deuterium atom from C-1 to C-2 was observed. These findings were confirmed by NMR analysis of the acetate obtained by fermentation of 2-phenoxy-[2-13 C,1-2 H 2 ]ethanol, 2-phenoxy-[1-13 C,1-2 H 2 ]ethanol, and 2-phenoxy-[1,2-13 C 2 ,1-2 H 2 ]ethanol. During the course of the biotransformation process, the molecular integrity of the glycolic unit was completely retained, no loss of the migrating deuterium occurred by exchange with the medium, and the 1,2deuterium shift was intramolecular. A diol dehydrataselike mechanism could explain the enzymatic cleavage of the ether bond of 2-phenoxyethanol, provided that an intramolecular H/OC 6 H 5 exchange is assumed, giving rise to the hemiacetal precursor of acetaldehyde. However, an alternative mechanism is proposed that is supported by the well recognized propensity of ␣-hydroxyradical and of its conjugate base (ketyl anion) to eliminate a ␤-positioned leaving group. Ether linkages are comparably stable, and their cleavage requires rather rigorous conditions. Such cleavage reactions represent challenges also to microbes and their enzymes, and this difficulty causes the relative stability of many ether compounds in nature (1). An important group of xenobiotic ether compounds, the linear polyether PEG 1 and its derivatives, is released into the * This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft, Bonn in its priority program "Radicals in enzymatic catalysis." The costs of publication of this article were defrayed in part by the payment of page charges.

Bioorganic Chemistry, 2021

γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as fl... more γ-Glutamyl derivatives of proteinogenic or modified amino acids raise considerable interest as flavor enhancers or biologically active compounds. However, their supply, on a large scale and at reasonable costs, remains challenging. Enzymatic synthesis has been recognized as a possible affordable alternative with respect to both isolation procedures from natural sources, burdened by low-yield and by the requirement of massive amount of starting material, and chemical synthesis, inconvenient because of the need of protection/deprotection steps. The E. coli γ-glutamyltransferase (Ec-GGT) has already been proposed as a biocatalyst for the synthesis of various γ-glutamyl derivatives. However, enzymatic syntheses using this enzyme usually provide the desired products in limited yield. Hydrolysis and autotranspeptidation of the donor substrate have been identified as the side reactions affecting the final yield of the catalytic process. In addition, experimental conditions need to be specifically adjusted for each acceptor substrate. Substrate specificity and the fine characterization of the activities exerted by the enzyme over time has so far escaped rationalization. In this work, reactions catalyzed by Ec-GGT between the γ-glutamyl donor glutamine and several representative acceptor amino acids have been finely analyzed with the identification of single reaction products over time. This approach allowed to rationalize the effect of donor/acceptor molar ratio on the outcome of the transpeptidation reaction and on the distribution of the different byproducts, inferring a general scheme for Ec-GGT-catalyzed reactions. The propensity to react of the different acceptor substrates is in agreement with recent findings obtained using model substrates and further supported by x-ray crystallography and will contribute to characterize the still elusive acceptor binding site of the enzyme.

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the... more Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investi-gated with a bioinformatics appro...