Glycosidase Activities Research Articles

Abstract P1172: Shear-mediated Remodeling Of Platelet Surface Glycosylation: How Can Platelets Trim Their Sugar Coats?

Mechanical circulatory support (MCS) is associated with bleeding complications often requiring multiple transfusions or reoperation. Hypershear stress within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, impaired aggregation, and microvesiculation - all contributors to bleeding. Recent studies showed that glycosylation of surface receptors, i.e. “ sugar coat ,” plays a major role in the regulation of platelet function and lifespan. We tested the hypothesis that shear stress promotes platelet surface deglycosylation by increasing platelet glycosidase activity. Methods: Human platelets were obtained from ACD-anticoagulated blood via gel filtration on Sepharose 2B. Platelets were exposed to neuraminidase and continuous shear stress in a hemodynamic shearing device. Platelets were stained with fluorophore-conjugated lectins binding terminal sialic acids (SNA I, MAL II) and galactose (ECA), anti-CD41 and anti-CD42a a/b. Multi-colored flow cytometry was used to quantify platelet surface glycosylation. Neuraminidase and galactosidase activities of intact and lysed platelets were measured using enzyme-specific fluorogenic substrates. Results: Exposure to shear stress and neuraminidase induced a significant decrease in platelet 2,6-sialylation and galactosylation, while 2,3-sialylation remained unaltered. Neuraminidase reinforced shear-mediated platelet desialylation, causing a prominent decrease in SNA binding even at low shear, and increased platelet galactose levels by unmasking the galactose residues capped by sialic acids. Shear stress resulted in a 9.5-fold increase in platelet neuraminidase and a 1.4-fold increase in galactosidase activities vs their basal levels. Neutral neuraminidase and galactosidase were upregulated by shear, while acidic isoforms did not increase, suggesting that other than lysosomal glycosidase pools are involved. Shear stress promotes platelet deglycosylation via upregulation of platelet neuraminidase and galactosidase activities. Developing therapeutic strategies for the preservation of platelet glycosylation offers significant translational potential for the pharmacologic management of MCS platelet dysfunction and bleeding.

Dynamic changes occur in the cell wall composition and related enzyme activities during flower development in Rosa damascena.

The flowering period of oil-bearing rose is short and many physiological processes occur during flower development. Changes in the cell wall composition and associated enzyme activities are important as they allow cells to divide, differentiate and grow. In the present study, changes in seven cell wall components and six cell wall-related enzyme activities at five flower development stages were investigated and the relationships between these parameters and flowering were examined. Ash content did not change between stages I to II but decreased at later stages. Neutral detergent fiber (NDF), acid detergent fiber (ADF) and hemicellulose contents increased after stage I but did not change significantly at the other developmental periods. Total pectin content increased throughout flower development. An "increase-decrease" trend was observed in total cellulose content and a "decrease-increase" pattern in uronic acid content. The activities of both glycosidases (β-galactosidase, β-glucosidase and endoglucanase) and pectinases (pectin lyase, pectin methyl esterase and polygalacturonase) increased until stage IV and decreased significantly at stage V of flower development. Correlation analysis revealed 14 positive and one negative correlation with the studied parameters. Cell wall enzymes showed positive correlations with each other. Principal component analysis (PCA) showed that ADF, NDF and cellulose content were significantly altered at stage II of flower development, and significant changes occurred in all cell wall enzyme activities between stages III and V. Overall, blooming is correlated closely with increased pectin and decreased cellulose contents, and changes in cell wall glucosidase and pectin hydrolysis enzyme activities. These results show that cell wall modifying enzymes are part of the flower development process in oil-bearing rose. Therefore, remodeling of cell wall components in petals is a process of flower development.

Synthesis and Structural Investigation of Foldamer-Based Iodine-Supported Artificial Glycosidases.

Glycosidases are a type of enzyme that hydrolytically cleave carbohydrates and form glycans for biologically important processes. The inadequacies of glycosidases or their genetic abnormalities are responsible for various diseases. Thus, the development of glycosidase mimetics is of great importance. We have designed and synthesized an enzyme mimetic containing l-phenylalanine, α-aminoisobutyric acid (Aib), l-leucine, and m-Nifedipine. From X-ray crystallography, the foldamer adopts a β-hairpin conformation stabilized by two 10-member and one 18-member NH⋅⋅⋅O=C hydrogen bonds. Moreover, the foldamer was found to be highly efficient in hydrolysing ethers and glycosides in the presence of iodine at room temperature. Further, X-ray analysis shows the backbone conformation of the enzyme mimetic to be almost unchanged after the glycosidase reaction. This is the first example of iodine-supported artificial glycosidase activity with an enzyme mimic at ambient conditions.

Radix Paeoniae Alba attenuates Radix Bupleuri-induced hepatotoxicity by modulating gut microbiota to alleviate the inhibition of saikosaponins on glutathione synthetase

Radix Bupleuri (RB) is commonly used to treat depression, but it can also lead to hepatotoxicity after long-term use. In many anti-depression prescriptions, RB is often used in combination with Radix Paeoniae Alba (RPA) as an herb pair. However, whether RPA can alleviate RB-induced hepatotoxicity remain unclear. In this work, the results confirmed that RB had a dose-dependent antidepressant effect, but the optimal antidepressant dose caused hepatotoxicity. Notably, RPA effectively reversed RB-induced hepatotoxicity. Afterward, the mechanism of RB-induced hepatotoxicity was confirmed. The results showed that saikosaponin A and saikosaponin D could inhibit GSH synthase (GSS) activity in the liver, and further cause liver injury through oxidative stress and nuclear factor kappa B (NF-κB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway. Furthermore, the mechanisms by which RPA attenuates RB-induced hepatotoxicity were investigated. The results demonstrated that RPA increased the abundance of intestinal bacteria with glycosidase activity, thereby promoting the conversion of saikosaponins to saikogenins in vivo. Different from saikosaponin A and saikosaponin D, which are directly combined with GSS as an inhibitor, their deglycosylation conversion products saikogenin F and saikogenin G exhibited no GSS binding activity. Based on this, RPA can alleviate the inhibitory effect of saikosaponins on GSS activity to reshape the liver redox balance and further reverse the RB-induced liver inflammatory response by the NF-κB/NLRP3 pathway. In conclusion, the present study suggests that promoting the conversion of saikosaponins by modulating gut microbiota to attenuate the inhibition of GSS is the potential mechanism by which RPA prevents RB-induced hepatotoxicity.

Insights into the regulation of cellular Mn2+ homeostasis via TMEM165

Golgi cation homeostasis is known to be crucial for many cellular processes including vesicular fusion events, protein secretion, as well as for the activity of Golgi glycosyltransferases and glycosidases. TMEM165 was identified in 2012 as the first cation transporter related to human glycosylation diseases, namely the Congenital Disorders of Glycosylation (CDG). Interestingly, divalent manganese (Mn) supplementation has been shown to suppress the observed glycosylation defects in TMEM165-deficient cell lines, thus suggesting that TMEM165 is involved in cellular Mn homeostasis. This paper demonstrates that the origin of the Golgi glycosylation defects arises from impaired Golgi Mn homeostasis in TMEM165-depleted cells. We show that Mn supplementation fully rescues the Mn content in the secretory pathway/organelles of TMEM165-depleted cells and hence the glycosylation process. Strong cytosolic and organellar Mn accumulations can also be observed in TMEM165- and SPCA1-depleted cells upon incubation with increasing Mn concentrations, thus demonstrating the crucial involvement of these two proteins in cellular Mn homeostasis. Interestingly, our results show that the cellular Mn homeostasis maintenance in control cells is correlated with the presence of TMEM165 and that the Mn-detoxifying capacities of cells, through the activity of SPCA1, rely on the Mn-induced degradation mechanism of TMEM165. Finally, this paper highlights that TMEM165 is essential in secretory pathway/organelles Mn homeostasis maintenance to ensure both Golgi glycosylation enzyme activities and cytosolic Mn detoxification.

Vinyl Halide‐Modified Unsaturated Cyclitols are Mechanism‐Based Glycosidase Inhibitors

AbstractSuitably configured allyl ethers of unsaturated cyclitols act as substrates of β‐glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of β‐glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter‐intuitive trend wherein the most electronegative substituents yielded the most labile pseudo‐glycosidic linkages. Structures of complexes with the Sulfolobus β‐glucosidase revealed similar enzyme‐ligand interactions to those seen in complexes with a 2‐fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7‐fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.

Vinyl Halide-Modified Unsaturated Cyclitols are Mechanism-Based Glycosidase Inhibitors.

Suitably configured allyl ethers of unsaturated cyclitols act as substrates of β-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of β-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages. Structures of complexes with the Sulfolobus β-glucosidase revealed similar enzyme-ligand interactions to those seen in complexes with a 2-fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7-fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.

Metabolomics and chemometrics depict the changes in the chemical profile of white lupine (Lupinus albus L.) bioactive metabolites during seed germination

The current study attempts to illustrate how the chemical and biological profile of white lupine seeds varies throughout the course of various germination days using UHPLC-QqQ-MS combined to chemometrics. Abscisic acid showed maximum level in the un-germinated seeds and started to decline with seed germination accompanied by an increase in the levels of gibberellins which were undetectable in un-germinated seeds. Coumaronochromones were the most prevalent constituents detected in un-germinated seeds while day 2 sprouts showed significant accumulation of flavones. The levels of alkaloids showed significant increase upon germination of the seeds reaching its maximum in day 14 sprouts. The OPLS model coefficients plot indicated that lupinalbin D and F, apigenin hexoside, kaempferol hexoside, albine, and hydoxylupanine showed strong positive correlation to the alpha amylase inhibitory activity of the tested samples while lupinalbin A, lupinisoflavone, lupinic acid and multiflorine were positively correlated to the inhibition of alpha glycosidase activity. The results obtained indicated that seed germination has a profound effect on the chemical profile as well as the in-vitro antidiabetic activity of lupine seeds.

Bacteria of the Black Sea are Producers of α-L-Rhamnosidase

The search for new producers of α-L-rhamnosidases and the study of their properties are constantly carried out, which makes it possible to identify enzymes with unique properties. Thus, the α-L-rhamnosidases producers of marine species of microorganisms can radically differ in habitat conditions from terrestrial ones. Previously, we have isolated the producers of α-L-rhamnosidases from a number of representatives of the Black Sea microbiota. However, the results of these studies did not allow us to isolate a strain promising for further study of the α-L-rhamnosidase synthesized by it. Therefore, the purpose of this work was to further search for effective producers of α-L-rhamnosidases among the microbiota of the Black Sea obtained from its different depths. Methods. Glycosidase activities were determined by the Romero and Davis methods. Results. The study of α-L-rhamnosidase activity in the dynamics of growth of 10 cultures isolated from the Black Sea showed that the only glycosidase activity, which was found on the third day of cultivation in six (07, 044, 050, 052, 054, 247) of ten cultures studied, was toward α-L-rhamnosidase. However, on the 5th day of cultivation, an increase (and in some strains, the appearance) of activity was noted in all tested cultures (from 0.01 to 0.12 U/mL). On the 7th day of cultivation, α-L-rhamnosidase activity in the supernatant of the culture liquid ranged from 0.02 to 0.2 U/mL. The highest activity (0.2 U/mL) was found in strain 052. On the 10th day of cultivation, the maximum activity (0.55 U/mL) was noted in culture 052 and slightly lower (0.35 U/mL, 0.28 U/mL, and 0.23 U/mL) in cultures 044, 050, and 054 respectively. Cultures 051, 020, and 247, which showed the same activity (0.1 U/mL), as well as 056 (0.09 U/mL) were an order of magnitude less active. The minimum activity was noted in culture 046 (0.03 U/mL). In the supernatant of the culture liquid of strain 07 on the 10th day of cultivation, α-L-rhamnosidase was absent at all. Since the highest α-L-rhamnosidase activity was found in the supernatants of culture liquids of 5 strains (044, 052, 054, 056, and 247), partially purified complex preparations of those α-L-rhamnosidases were obtained for further research. The study of the substrate specificity of complex enzyme preparations of α-L-rhamnosidases of strains 044, 051, 052, 056, and 247 on natural flavonoids, such as naringin, neohesperidin, and rutin, indicated that α-L-rhamnosidase obtained from strain 052 showed the highest activity on three investigated substrates: rutin, naringin (0.55 U/mL), and neohesperidin (0.52 U/mL). In addition to natural substrates, complex preparations of α-L-rhamnosidases of strains 052, 054, 056, and 247 also hydrolyzed synthetic derivatives of monosaccharides, such as p-nitrophenyl-α-L-rhamnopyranoside and p-nitrophenyl-β-D-glucopyranoside. The maximum activity (0.15 U/mL) was noted in strain 052, whereas strain 044 was unable to hydrolyze synthetic substrates. Conclusions. Complex enzyme preparations of α-L-rhamnosidase obtained from strain 052 are promising for further investigations. They showed the highest activity both on three natural substrates, such as rutin, naringin, and neohesperidin, as well as on such synthetic derivatives of monosaccharides as p-nitrophenyl-α-L-rhamnopyranoside and p-nitrophenyl-β-D-glucopyranoside.

Masters of Misdirection: Peptidoglycan Glycosidases in Bacterial Growth.

The dynamic composition of the peptidoglycan cell wall has been the subject of intense research for decades, yet how bacteria coordinate the synthesis of new peptidoglycan with the turnover and remodeling of existing peptidoglycan remains elusive. Diversity and redundancy within peptidoglycan synthases and peptidoglycan autolysins, enzymes that degrade peptidoglycan, have often made it challenging to assign physiological roles to individual enzymes and determine how those activities are regulated. For these reasons, peptidoglycan glycosidases, which cleave within the glycan strands of peptidoglycan, have proven veritable masters of misdirection over the years. Unlike many of the broadly conserved peptidoglycan synthetic complexes, diverse bacteria can employ unrelated glycosidases to achieve the same physiological outcome. Additionally, although the mechanisms of action for many individual enzymes have been characterized, apparent conserved homologs in other organisms can exhibit an entirely different biochemistry. This flexibility has been recently demonstrated in the context of three functions critical to vegetative growth: (i) release of newly synthesized peptidoglycan strands from their membrane anchors, (ii) processing of peptidoglycan turned over during cell wall expansion, and (iii) removal of peptidoglycan fragments that interfere with daughter cell separation during cell division. Finally, the regulation of glycosidase activity during these cell processes may be a cumulation of many factors, including protein-protein interactions, intrinsic substrate preferences, substrate availability, and subcellular localization. Understanding the true scope of peptidoglycan glycosidase activity will require the exploration of enzymes from diverse organisms with equally diverse growth and division strategies.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural β-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and β-anomeric linkages, N-glycosides with β-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Synthesis, in silico, in vitro and in vivo studies of novel natural-based arylidenes curcumin as potential glycohydrolase digestive enzymes inhibitors

Diabetes is one of the most common metabolic diseases in humans and the use of herbal medicines is of great clinical importance to inhibit carbohydrate-hydrolyzing enzymes and reduce blood glucose levels in diabetic patients. Inhibition of glycosidase activity is an effective way to treat and prevent diabetes. Therefore, in this study, curcumin-based benzaldehyde derivatives were synthesized and used as influential agents in the treatment of diabetes with inhibitory properties against two carbohydrate-hydrolyzing enzymes α-glucosidase (α-Glu) and α-amylase (α-Amy) as significant therapeutic targets for reducing postprandial hyperglycemia. Overall, the findings showed that due to the specific inhibitory activity against α-Glu in comparison with α-Amy, as well as more stability and antioxidant activity than curcumin, C5 and C8 derivatives are potentially important anti-diabetic drugs, not only to decrease glycemic index but also to limit the activity of the main production pathways of reactive oxygen species (ROS) in diabetic patients. Communicated by Ramaswamy H. Sarma

Ecophysiological Properties and Hydrolytic Activity of Chemoorganotrophic Bacteria from Holosiivskyi National Nature Park

Any natural ecosystem contains a specific range of microorganisms. The anthropogenic impact can cause a change in the growth conditions of soil and rhizospheric microbiome and affect the number and the physiological properties of microorganisms. The aim of the study was to isolate the representative microorganisms from terrestrial ecosystems of Holosiivskyi National Nature Park (Ukraine) that are not exposed to extreme factors, to study their ecophysiological properties (resistance to UV radiation, dehydration, hypersalinity, temperature), and to study the extracellular glycoside and proteolytic activities. Methods. Aerobic chemoorganotrophic bacteria isolated at 30°C from soil and phytocenoses of Holosiivskyi National Nature Park were studied. Meat-peptone agar was used to cultivate bacteria. Bacterial UV irradiation was performed with a BUF-15 lamp (λ=254 nm) in the range of 30—1350 J/m2. The temperature range of growth and halotolerance of microorganisms was determined in the range of 1—42°C and 0.1—150 g NaCl/L, respectively. Bacterial isolates were cultivated in submerged conditions at 28°C for 4 days. Synthetic p-nitrophenyl substrates, soluble starch, and guar galactomannan were used to determine glycosidase activity. To study proteolytic activity, casein, elastin, and gelatin were used. Results. The study of 14 soil and plant samples revealed the number of bacteria detected from 9.3×104 to 4.8×105 CFU/g in winter, and 4.8×105 to 4.2×106 CFU/g in summer. The microorganisms were represented by 1—4 morphotypes. There were isolated 37 isolates of aerobic chemoorganotrophic microorganisms, and 69% of them were represented by gram-positive rods. Th e dominance of pigmented isolates was not detected. Most of the microorganisms studied were psychrotolerant and moderate halophiles. The isolates 3g3, 8g1, 8g2, 8g3 from chornozem and dark gray soil showed high resistance to UV radiation. Th e LD99.99 ranged from 800 to 1100 J/m2. The isolates from chornozem, birch moss, green moss with sand and soil, and green moss from oak (1g, 4g2, 9g1, 14g2) were moderately resistant. The LD99.99 was 280—650 J/m2. The UV resistance was shown to be independent of pigmentation. It correlated with dehydration. The phenomenon of resistance to such UV radiation and dehydration may indicate the presence of active reparation mechanisms of DNA damage. All isolates showed cellulose and hemicellulose degrading activities as well as caseinolytic activity. Isolate 9g1 showed high β-xylosidase activity. Conclusions. The high resistance to UV radiation and dehydration of non-adapted microorganisms as well as the wide range of exohydrolase activity indicate the wide adaptive capacity of microorganisms from natural ecosystems, which goes beyond the influence of surrounding factors. No data existed in the available literature defining hydrolytic activity and resistance of microorganisms of the temperate region of Ukraine to extreme factors. The obtained experimental data will allow for a better understanding of the resistance level of microorganisms of temperate regions to extreme factors. As a result of the work, new bacteria with high degrading activity were isolated. The studied isolates require further characterization and analysis for biotechnological applications.

Enhancing the Production of Hydrolytic Enzymes in Elicited Tamarillo (Solanum betaceum Cav.) Cell Suspension Cultures.

Plant cell suspension cultures are widely used as a tool for analyzing cellular and molecular processes, metabolite synthesis, and differentiation, bypassing the structural complexity of plants. Within the range of approaches used to increase the production of metabolites by plant cells, one of the most recurrent is applying elicitors capable of stimulating metabolic pathways related to defense mechanisms. Previous proteomics analysis of tamarillo cell lines and cell suspension cultures have been used to further characterize and optimize the growth and stress-related metabolite production under in vitro controlled conditions. The main objective of this work was to develop a novel plant-based bioreactor system to produce hydrolytic enzymes using an elicitation approach. Based on effective protocols for tamarillo micropropagation and plant cell suspension culture establishment from induced callus lines, cell growth has been optimized, and enzymatic activity profiles under in vitro controlled conditions characterized. By testing different sucrose concentrations and the effects of two types of biotic elicitors, it was found that 3% (w/v) sucrose concentration in the liquid medium enhanced the production of hydrolytic enzymes. Moreover, casein hydrolysate at 0.5 and 1.5 g/L promoted protein production, whereas yeast extract (0.5 g/L) enhanced glycosidase activity. Meanwhile, chitosan (0.05 and 0.1 g/L) enhanced glycosidases, alkaline phosphates, and protease activities.

Improving the catalytic ability of a peptide-based artificial glycosidase through a tyrosine strategy.

Recently, peptide-based artificial enzymes have attracted growing interest due to the similar composition of peptide assemblies to natural enzymes. However, low catalytic activity and stability are still the main challenges for these enzyme-like catalysts. In this study, a tyrosine strategy was developed to construct a peptide-based artificial glycosidase through engaging the Tyr residue into the peptide sequence. We found that the appropriate substitution of Tyr produced an enhanced catalytic ability, because the proximal Tyr residue around catalytic sites acts as a nucleophile, playing an important role in the substrate orientation and proton transfer. Moreover, inspired by the biological function of dityrosine bonds in structural proteins, a photo-crosslinking reaction between the phenolic side chains of Tyr was also employed to form intermolecular covalent bonds in peptide assemblies, which further improved the activity and stability of the artificial glycosidase. This work provides a simple avenue for the designing and construction of efficient glycosidase-like catalysts based on a peptide-based material platform.

Analysis of Glycosidase Activities, Steroids Hormones Concentrations and Selected Biochemical Parameters in Serum of Dromedary Camels (Camelus dromedarius) during Follicular Phase and Parturition

Analysis of Glycosidase Activities, Steroids Hormones Concentrations and Selected Biochemical Parameters in Serum of Dromedary Camels (Camelus dromedarius) during Follicular Phase and Parturition

Glycosidase-targeting small molecules for biological and therapeutic applications.

Glycosidases are ubiquitous enzymes that catalyze the hydrolysis of glycosidic linkages in oligosaccharides and glycoconjugates. These enzymes play a vital role in a wide variety of biological events, such as digestion of nutritional carbohydrates, lysosomal catabolism of glycoconjugates, and posttranslational modifications of glycoproteins. Abnormal glycosidase activities are associated with a variety of diseases, particularly cancer and lysosomal storage disorders. Owing to the physiological and pathological significance of glycosidases, the development of small molecules that target these enzymes is an active area in glycoscience and medicinal chemistry. Research efforts carried out thus far have led to the discovery of numerous glycosidase-targeting small molecules that have been utilized to elucidate biological processes as well as to develop effective chemotherapeutic agents. In this review, we describe the results of research studies reported since 2018, giving particular emphasis to the use of fluorescent probes for detection and imaging of glycosidases, activity-based probes for covalent labelling of these enzymes, glycosidase inhibitors, and glycosidase-activatable prodrugs.

Ratiometric sensing of β-galactosidase based on excited-state intramolecular proton transfer (ESIPT) and solid-state luminescence enhancement

An ESIPT-based ratiometric probe with solid-state luminescence enhancement is developed for the sensitive detection of glycosidase activity.

Analysis of the oral microbiome during hormonal cycle and its alterations in menopausal women: the “AMICA” project

The maintenance of human health is dependent on a symbiotic relationship between humans and associated bacteria. The diversity and abundance of each habitat’s signature microbes vary widely among body areas and among them the oral microbiome plays a key role. Significant changes in the oral cavity, predominantly at salivary and periodontal level, have been associated with changes in estrogen levels. However, whether the oral microbiome is affected by hormonal level alterations is understudied. Hence the main objective pursued by AMICA project was to characterize the oral microbiome (saliva) in healthy women through: profiling studies using "omics" technologies (NMR-based metabolomics, targeted lipidomics by LC–MS, metagenomics by NGS); SinglePlex ELISA assays; glycosidase activity analyses and bioinformatic analysis. For this purpose, thirty-nine medically healthy women aged 26–77 years (19 with menstrual cycle and 20 in menopause) were recruited. Participants completed questionnaires assessing detailed medical and medication history and demographic characteristics. Plasmatic and salivary levels of sexual hormones were assessed (FSH, estradiol, LH and progesteron) at day 3 and 14 for women with menstrual cycle and only once for women in menopause. Salivary microbiome composition was assessed through meta-taxonomic 16S sequencing and overall, the salivary microbiome of most women remained relatively stable throughout the menstrual cycle and in menopause. Targeted lipidomics and untargeted metabolomics profiling were assessed through the use of LC–MS and NMR spectroscopy technologies, respectively and significant changes in terms of metabolites were identified in saliva of post-menopausal women in comparison to cycle. Moreover, glycosyl hydrolase activities were screened and showed that the β-D-hexosaminidase activity was the most present among those analyzed. Although this study has not identified significant alterations in the composition of the oral microbiome, multiomics analysis have revealed a strong correlation between 2-AG and α-mannosidase. In conclusion, the use of a multidisciplinary approach to investigate the oral microbiome of healthy women provided some indication about microbiome-derived predictive biomarkers that could be used in the future for developing new strategies to help to re-establish the correct hormonal balance in post-menopausal women.

Investigation of non-Saccharomyces yeasts with intracellular β-glycosidase activity for wine aroma modification.

Since high proportions of aroma-relevant molecules in plant-derived juices are present in glycosylated forms, the introduction of glycosidase activity during processing is an important tool to modify the aroma composition of the product. During winemaking, the addition of β-glycosidase enzyme or microorganisms with β-glycosidase activity is an established technology. However, low stability under acidic conditions and low selectivity for hydrolysis of different glycosides are still drawbacks, which limit application possibilities. Here, we report the identification and characterization of non-Saccharomyces yeast strains with relatively high β-glycosidase activity in their cultures. We found strong indications for intracellular localization of the enzymes, which is in line with the pH robustness found in experiments with whole cells. Furthermore, we compared the selectivity of aroma compound release from glycoside mixtures using whole cells or cell extracts. The results showed strong differences for the released aroma patterns, which indicates the transport of glycosides and intracellular hydrolysis. Our work demonstrates the application potential of yeasts with intracellular β-glycosidase activities as catalysts with high pH robustness and selective aroma release properties. PRACTICAL APPLICATION: The yeast strains identified and characterized within this work can be applied in wine processing but also in other processes to release aroma molecules from their glycosylated precursors provided by the plants. The strains show relatively high activity of the relevant enzyme, β-glycosidase, also at low pH, which is essential in many processes. In contrast to most other approaches, the enzyme is inside the cells, which can lead to a specific release of certain aroma compounds.