Hydroxyapatite: An Eco-Friendly Material for Enzyme Immobilization.

Biocatalysis has emerged in the last decade as a valuable and eco-friendly tool in chemical synthesis, allowing in several instances to reduce or eliminate the use of hazardous reagents, environmentally dangerous solvents and harsh reaction conditions. Enzymes are indeed able to catalyse chemical transformations on non-natural substrates under mild reaction conditions, still maintaining their high chemo-, regio-, and stereoselectivity. Enzyme immobilization, i. e. the grafting of enzymes on solid supports, can be viewed as an enabling technology, as it allows a better control of the reaction and the recycling of the biocatalyst, thus rendering economically viable the use of expensive enzymes also on a large scale. To pursue a sustainable approach, the supports for enzyme immobilization should be eco-friendly and possibly renewable. This review highlights the use of hydroxyapatite (HAP), an inorganic biomaterial able to confer strength and stiffness to the bone tissue in animals, as carrier for enzyme immobilization. HAP is a cheap, non-toxic and biocompatible material, with high surface area and protein affinity. Different enzyme classes, immobilization strategies, and the use of diverse HAP-based supports will be discussed, underlining the immobilization conditions and the properties of the obtained biocatalysts.

l-Theanine Goes Greener: A Highly Efficient Bioprocess Catalyzed by the Immobilized γ-Glutamyl Transferase from Bacillus subtilis.

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.

Preparation, Characterization and In Vitro Stability of a Novel ACE-Inhibitory Peptide from Soybean Protein.

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 < 1 kDa.

From Batch to Continuous Flow Bioprocessing: Use of an Immobilized γ-Glutamyl Transferase from B. subtilis for the Synthesis of Biologically Active Peptide Derivatives.

γ-Glutamyl-peptides are frequently endowed with biological activities. In this work, "kokumi peptides" such as γ-glutamyl-methionine (1) and γ-glutamyl-(S)-allyl-cysteine (2), as well as the neuroprotective γ-glutamyl-taurine (3) and the antioxidant ophthalmic acid (4), were synthesized through an enzymatic transpeptidation reaction catalyzed by the γ-glutamyl transferase from Bacillus subtilis (BsGGT) using glutamine as the γ-glutamyl donor. BsGGT was covalently immobilized on glyoxyl-agarose resulting in high protein immobilization yield and activity recovery (>95%). Compounds 1-4 were obtained in moderate yields (19-40%, 5-10 g/L) with a variable purity depending on the presence of the main byproduct (γ-glutamyl-glutamine, 0-16%). To achieve process intensification and better control of side reactions, the synthesis of 2 was moved from batch to continuous flow. The specific productivity was 1.5 times higher than that in batch synthesis (13.7 µmol/min*g), but it was not accompanied by a paralleled improvement of the impurity profile.

An overall framework for the E. coli γ-glutamyltransferase-catalyzed transpeptidation reactions.

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.

Determination of flavor-potentiating compounds in different Italian tomato varieties.

Umami taste, known as appetizing sensation, is mainly imparted by monosodium glutamate (MSG, the first identified umami factor) in synergistic combination with some 5' ribonucleotides such as inosine 5'-monophosphate, IMP, guanosine 5'-monophosphate, GMP, and adenosine 5'-monophoshate, AMP. The level of free glutamic acid in tomatoes is higher than in other vegetables or fruits and increases with ripening and industrial processing. In addition, due to the presence of bioactive metabolites, tomatoes and tomato-based products are among the most consumed healthy food items. The levels of the major umami compounds of tomato, that is, glutamate and 5'-ribonucleotides (GMP and AMP) were assessed in different parts (skin, outer flesh, and inner pulp) of known tomato varieties from southern Italy: San Marzano Originale, San Marzano 245, Black Tomato, Corbarino Corbara, Corbarino Nocera, and Superpomodoro (tomato hybrid). Such varieties were also investigated for their antioxidant properties through DMPD, DPPH, and ABTS assays, with San Marzano Originale showing the highest antioxidant power both in lipophilic and methanolic fractions. The concentration of umami compounds in tomato differs with the part of the fruit analyzed and is greatly dependent on the variety, being Corbarino Nocera the cultivar richest in glutamate and Superpomodoro in ribonucleotides. As for nutritional aspect, results confirm the great nutraceutical feature of San Marzano tomato, the most known variety used in industrial processes. PRACTICAL APPLICATIONS: This study was planned to develop a method to quantify the major umami compounds that strongly influence the organoleptic properties of many different tomato varieties. It is known that the sensory quality of fruits and vegetables is an important factor in consumer's choice. The analytical methods described here enabled the evaluation of the glutamate and 5'-ribonucleotides contents in six selected varieties of tomato from Campania region, and can be easily used to determine the sensory profile of commercial varieties, for example, those perceived as not very tasteful by consumers.

Simulating Multiple Substrate-Binding Events by γ-Glutamyltransferase Using Accelerated Molecular Dynamics.

γ-Glutamyltransferase (GGT) is an enzyme that uses γ-glutamyl compounds as substrates and catalyzes their transfer to a water molecule or an acceptor substrate with varied physiological function in bacteria, plants, and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, the structural dynamics of the substrate binding to the catalytic site of GGT are unknown. Here, we modeled Escherichia coli GGT's glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggest its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.

Influence of drying techniques and growing location on the chemical composition of sweet pepper (Capsicum annuum L., var. Senise).

The aim of this work was the comprehensive characterization of dried Senise pepper, a tasty landrace, to verify the suitability of common industrial processing and different growing locations to its production. Fruits from experimental CREA fields of Battipaglia (southern Italy) and Montanaso (northern Italy), dried using a solar dryer or a forced air oven, were compared with lyophilized and commercial samples. Solar dried samples showed a retention of glucose, while fructose levels decreased. Citric acid was the main acid in all samples, showing a differentiated behavior upon processing, with higher content in Battipaglia samples. The ascorbic acid showed the highest correlation with antioxidant assays, and both drying methods decreased its content, with better retention in Battipaglia samples. Drying did not markedly affect carotenoid levels, showing higher content in Battipaglia peppers. Both drying technique and mostly growing location influenced the volatile profile, with higher apocarotenoid content in Battipaglia samples. PRACTICAL APPLICATIONS: The use of Senise red pepper, a tasty landrace awarded in 1996 with the PGI certification mark, is spreading even outside Italy due to the renewed interest for high quality products with excellent taste and healthy properties. These peppers are mostly sun-dried following a traditional procedure and consumed as spice. New approaches regarding the type of drying and the growing location of this spice, together with the knowledge about the changes in quality indexes with processing, can be useful for a better exploitation of this product, increasing its market availability and lowering the cost of production.

Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant B. subtilis γ-glutamyltransferase (GGT).

γ-Glutamylpeptides are compounds derived from the acylation of an amino acid or a short peptide by the γ-carboxyl carbon of the side chain of glutamic acid. Due to their altered chemico-physical and organoleptic properties, they may be interesting substitutes or precursors of parent compounds used in pharmaceutical, dietetic and cosmetic formulations. Some of them are naturally occurring flavor enhancers or are endowed with biological activities. Enzymatic approaches to the synthesis of γ-glutamyl derivatives based on the use of γ-glutamyltransferases (GGTs, EC 2.3.2.2) have been proposed, which should be able to alleviate the problems connected with the troublesome and low-yielding extraction from natural sources or the non-economical chemical synthesis, which requires protection/deprotection steps. With the aim of overcoming the current limitations in the use of GGTs as biocatalysts, a mutant GGT was investigated. The mutant GGT was obtained by inserting the active-site-covering lid loop of the E. coli GGT onto the structure of B. subtilis GGT. With respect to the wild-type enzyme, the mutant showed a more demanding substrate specificity and a low hydrolase activity. These results represent an attempt to correlate the structural features of a GGT to its different activities. However, the ability of the mutant enzyme to catalyze the subsequent addition of several γ-glutamyl units, inherited by the parent B. subtilis GGT, still represents a limitation to its full application as a biocatalyst for preparative purposes.

Data for the synthesis of oligo-γ-glutamylglutamines as model compounds for γ-glutamyltransferases (GGTs) and for normalization of activities of different GGTs.

γ-Glutamyltransferases (GGTs) are widespread, conserved enzymes that catalyze the transfer of the γ-glutamyl moiety from a donor substrate to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate. Although the vast majority of the known GGTs has a short sequence called lid-loop covering the glutamate binding site, Bacillus subtilis GGT and some other enzymes from Bacillus spp. lack the lid loop. In order to assess the possible role of the lid loop of GGTs in substrate selection, synthetic oligo-γ-glutamylglutamines containing up to three γ-glutamyl residues were used as model substrates. The activities of the enzymes under investigation were standardized with respect to a common reaction to ensure comparable results. The activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from Escherichia coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT (Calvio et al., 2018) [1]. Here we report the experimental procedures for the synthesis of model substrates γ-glutamylglutamines through the method of the N-phtaloyl-L-glutamic acid anhydride and the spectral data of the synthetized compounds. The data obtained in the normalization procedure of the activities of the three enzymes are also reported.

The structure of PghL hydrolase bound to its substrate poly-γ-glutamate.

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-γ-glutamic acid (γ-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 γ-PGA hydrolases, which can dismantle the γ-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 γ-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 γ-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.

Evidences on the role of the lid loop of γ-glutamyltransferases (GGT) in substrate selection.

γ-Glutamyltransferase (GGT) catalyzes the transfer of the γ-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate. The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lid-loop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-γ-glutamic acid (γ-PGA) as a substrate. Starting from this observation, in this work the activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT. Results indicate that the absence of the lid loop seems not to be the sole structural feature responsible for the recognition of a polymeric substrate by GGTs. Nevertheless, time course of hydrolysis reactions carried out using oligo-γ-glutamyl glutamines as substrates showed that the lid loop acts as a gating structure, allowing the preferential selection of the small glutamine with respect to the oligomeric substrates. In this respect, the mutant B. subtilis GGT revealed to be more similar to E. coli GGT than to its wild-type counterpart. In addition, the transpeptidase activity of the newly produced mutant enzyme revealed to be higher with respect to that of both E. coli and wild-type B. subtilis GGT. These findings can be helpful in selecting GGTs intended as biocatalysts for preparative purposes as well as in designing mutant enzymes with improved transpeptidase activity.

New ACE-Inhibitory Peptides from Hemp Seed (Cannabis sativa L.) Proteins.

A hemp seed protein isolate, prepared from defatted hemp seed meals by alkaline solubilization/acid precipitation, was subjected to extensive chemical hydrolysis under acid conditions (6 M HCl). The resulting hydrolysate was fractionated by semipreparative RP-HPLC, and the purified fractions were tested as inhibitors of angiotensin converting enzyme (ACE). Mono- and bidimensional NMR experiments and LC-MS analyses led to the identification of four potentially bioactive peptides, i.e. GVLY, IEE, LGV, and RVR. They were prepared by solid-phase synthesis, and tested for ACE-inhibitory activity. The IC50 values were GVLY 16 ± 1.5 µM, LGV 145 ± 13 µM, and RVR 526 ± 33 µM, confirming that hemp seed may be a valuable source of hypotensive peptides.

γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

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 investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Chemistry of α-mangostin. Studies on the semisynthesis of minor xanthones from Garcinia mangostana.

α-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 α-mangostin and describes the semisynthesis of some minor xanthones isolated from G. mangostana.

Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications.

We described the development of a biochromatographic system which uses a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) for the evaluation of the substrate specificity on nucleoside libraries. AhPNP has been covalently immobilized on a fused silica Open Tubular Capillary (OTC) via Schiff base chemistry. The resulting bioreactor has been characterized by the determination of kinetic constants (Km and Vmax) for a natural substrate (inosine) and then assayed versus all natural purine (deoxy)ribonucleosides and a small library of 6-substituted purine ribosides. Characterization of the bioreactor has been carried out through a bidimensional chromatographic system with the sample on-line transfer from the bioreactor to the analytical column for the separation and quantification of substrate and product. Comparison with the soluble enzyme showed that the AhPNP-based bioreactor is reliable as the same ranking order, with respect to the standard activity assay, was obtained. The stability of the IMER was also assessed and the system was found to be stable up to 60 reactions.

Characterisation of mucilages extracted from seven Italian cultivars of flax.

The chemical composition, physicochemical, functional and sensory properties of mucilages, extracted from seven Italian flax cultivars, were evaluated. All samples were composed of neutral and acidic sugars, with a low protein content. From the NMR data, a rhamnogalacturonan backbone could be inferred as a common structural feature for all the mucilages, with some variations depending on the cultivar. All the suspensions showed a poor stability, which was consistent with a low zeta potential absolute value. The viscosity seemed to be positively correlated with the neutral sugars and negatively with the amount of proteins. Functional properties were dependent on the cultivar. The sensory analysis showed that most mucilages are tasteless. All these outcomes could support the use of flaxseed mucilages for industrial applications. In particular, Solal and Festival cultivars could be useful as thickeners, due to their high viscosity, while Natural, Valoal and Kaolin as emulsifiers for their good surface-active properties.

pH-dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ-glutamyltransferase.

γ-Glutamyltransferases (γ-GTs) are heterodimeric enzymes that catalyze the transfer of a γ-glutamyl group from a donor species to an acceptor molecule in a transpeptidation reaction through the formation of an intermediate γ-glutamyl enzyme. In our search for a γ-GT from a generally recognized as safe microorganism suitable for the production of γ-glutamyl derivatives with flavor-enhancing properties intended for human use, we cloned and overexpressed the γ-GT from Bacillus subtilis. In this study, we report the behavior of B. subtilis γ-GT in reactions involving glutamine as the donor compound and various acceptor amino acids. The common thread emerging from our results is a strong dependence of the hydrolase, transpeptidase and autotranspeptidase activities of B. subtilis γ-GT on pH, also in relation to the pKa of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor molecule, undergoes rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to four γ-glutamyl moieties are linked to a single glutamine. Moreover, we found that D-glutamine is also recognized both as a donor and as an acceptor substrate. Our results prove that the B. subtilis γ-GT-catalyzed transpeptidation reaction is feasible, and the observed activities of γ-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material γ-polyglutamic acid.

Studies on umami taste. Synthesis of new guanosine 5'-phosphate derivatives and their synergistic effect with monosodium glutamate.

A number of N2-alkyl and N2-acyl derivatives of guanosine 5'-phosphate (GMP) have been synthesized and tested for their synergistic effect with monosodium L-glutamate (MSG), the prototypical substance imparting umami taste to savory-based foods. Capacities to enhance the taste intensity of MSG (gamma values) were estimated through subjective comparisons of MSG/nucleotide mixtures in water with appropriate solutions of MSG alone. Assuming beta = gamma[nucleotide]/gamma[IMP], beta values of the N2-substituted GMPs were found in the range 1.2-5.7. Such values appear to be related to the chain length of the substituent in the 2-position of the purine nucleus and dependent on the replacement of a CH 2 group with an S atom and/or with an alpha-CO group. These findings indicate that the exocyclic NHR group of the guanine moiety is actively implicated in the synergism between GMP derivatives and MSG. Theoretical calculations suggest that an anti conformation is probably assumed by ribonucleotide molecules interacting with umami receptors.

Mechanism of anaerobic degradation of triethanolamine by a homoacetogenic bacterium.

Triethanolamine (TEA) is converted into acetate and ammonia by a strictly anaerobic, gram-positive Acetobacterium strain LuTria3. Fermentation experiments with resting cell suspensions and specifically deuterated substrates indicate that in the acetate molecule the carboxylate and the methyl groups correspond to the alcoholic function and to its adjacent methylene group, respectively, of the 2-hydroxyethyl unit of TEA. A 1,2 shift of a hydrogen (deuterium) atom from -CH2-O- to =N-CH2- without exchange with the medium was observed. This fact gives evidence that a radical mechanism occurs involving the enzyme and/or coenzyme molecule as a hydrogen carrier. Such a biodegradation appears analogous to the conversion of 2-phenoxyethanol into acetate mediated by another strain of the anaerobic homoacetogenic bacterium Acetobacterium.