Gyöngyi Gyémánt

Action pattern and subsite mapping of Bacillus licheniformis α-amylase (BLA) with modified maltooligosaccharide substrates

This study represents the first characterisation of the substrate‐binding site of Bacillus licheniformis α‐amylase (BLA). It describes the first subsite map, namely, number of subsites, apparent subsite energies and the dual product specificity of BLA. The product pattern and cleavage frequencies were determined by high‐performance liquid chromatography, utilising a homologous series of chromophore‐substituted maltooligosaccharides of degree of polymerisation 4–10 as model substrates. The binding region of BLA is composed of five glycone, three aglycone‐binding sites and a ‘barrier’ subsite. Comparison of the binding energies of subsites, which were calculated with a computer program, shows that BLA has similarity to the closely related Bacillus amyloliquefaciens α‐amylase.

Subsite mapping of human salivary α-amylase and the mutant Y151M

This study characterizes the substrate‐binding sites of human salivary α‐amylase (HSA) and its Y151M mutant. It describes the first subsite maps, namely, the number of subsites, the position of cleavage sites and apparent subsite energies. The product pattern and cleavage frequencies were determined by high‐performance liquid chromatography, utilizing a homologous series of chromophore‐substituted maltooligosaccharides of degree of polymerization 3–10 as model substrates. The binding region of HSA is composed of four glycone and three aglycone‐binding sites, while that of Tyr151Met is composed of four glycone and two aglycone‐binding sites. The subsite maps show that Y151M has strikingly decreased binding energy at subsite (+2), where the mutation has occurred (−2.6 kJ/mol), compared to the binding energy at subsite (+2) of HSA (−12.0 kJ/mol).

Examination of the active sites of human salivary α-amylase (HSA)

The action pattern of human salivary amylase (HSA) was examined by utilising as model substrates 2-chloro-4-nitrophenyl (CNP) β-glycosides of maltooligosaccharides of dp 4–8 and some 4-nitrophenyl (NP) derivatives modified at the nonreducing end with a 4,6-O-benzylidene (Bnl) group. The product pattern and cleavage frequency were investigated by product analysis using HPLC. The results revealed that the binding region in HSA is longer than five subsites usually considered in the literature and suggested the presence of at least six subsites; four glycone binding sites (−4, −3, −2, −1) and two aglycone binding sites (+1, +2). In the ideal arrangement, the six subsites are filled by a glucosyl unit and the release of maltotetraose (G4) from the nonreducing end is dominant. The benzylidene group was also recognisable by subsites (−3) and (−4). The binding modes of the benzylidene derivatives indicated a favourable interaction between the Bnl group and subsite (−3) and an unfavourable one with subsite (−4). Thus, subsite (−4) must be more hydrophylic than hydrophobic. As compared with the action of porcine pancreatic α-amylase (PPA) on the same substrates, the results showed differences in the three-dimensional structure of active sites of HSA and PPA.

Evidence for pentagalloyl glucose binding to human salivary α-amylase through aromatic amino acid residues

We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K(EI)=2.6 microM and K(ESI)=3.9 microM were determined from Lineweaver-Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction.

“Back to the Future”: A New Look at Hydroxypropyl Beta-Cyclodextrins

Since the discovery about 30 years ago (2-hydroxypropyl) beta-cyclodextrin, a highly soluble derivative of beta-cyclodextrin, has become an approved excipient of drug formulations included both in the United States and European Pharmacopoeias. It is recommended to use as solubilizer and stabilizer for oral and parenteral formulations. Recently, its pharmacological activity has been recognized in various diseases. The increasing applications require a closer look to the structure-activity relationship. As (2-hydroxypropyl) beta-cyclodextrin (HPBCD) is always a mixture of isomers with various degrees and pattern of hydroxypropylation, no wonder that the products of different manufacturers are often different. Several HPBCDs were compared applying a battery of analytical tools including thin layer chromatography, high performance liquid chromatography (HPLC), HPLC-mass spectrometry (MS), and matrix-assisted laser desorption MS. We studied how the average degree of substitution affects the aggregation behavior, the toxicity, and the solubilizing effect on poorly soluble drugs. We found that the products with low average degree of substitution are more prone to aggregation. The samples studied are nontoxic to Caco-2 cells and have low hemolytic activity. The solubility enhancement of poorly soluble drugs decreases or increases with increasing degree of substitution or shows a maximum curve depending on the properties of the guest.

Mapping of barley α-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley α‐amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3–12, revealed unfavorable binding energies at the internal subsites −3 and −5 and at subsites −8 and +3/+4 defining these subsites as binding barriers. Barley α‐amylase 1 mutants Y105A and T212Y at subsite −6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high‐affinity subsites (−2 and +2) and barriers. The double mutant Y105A‐T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in α‐amylases.

Chemoenzymatic synthesis of 2-chloro-4-nitrophenyl β-maltoheptaoside acceptor-products using glycogen phosphorylase b

In the present work, we aimed at developing a chemoenzymatic procedure for the synthesis of beta-maltooligosaccharide glycosides. The primer in the enzymatic reaction was 2-chloro-4-nitrophenyl beta-maltoheptaoside (G(7)-CNP), synthesised from beta-cyclodextrin using a convenient chemical method. CNP-maltooligosaccharides of longer chain length, in the range of DP 8-11, were obtained by a transglycosylation reaction using alpha-D-glucopyranosyl-phosphate (G-1-P) as a donor. Detailed enzymological studies revealed that the conversion of G(7)-CNP catalysed by rabbit skeletal muscle glycogen phosphorylase b (EC 2.4.1.1) could be controlled by acarbose and was highly dependent on the conditions of transglycosylation. More than 90% conversion of G(7)-CNP was achieved through a 10:1 donor-acceptor ratio. Tranglycosylation at 37 degrees C for 30 min with 10 U enzyme resulted in G(8-->12)-CNP oligomers in the ratio of 22.8, 26.6, 23.2, 16.5, and 6.8%, respectively. The reaction pattern was investigated using an HPLC system. The preparative scale isolation of G(8-->11)-CNP glycosides was achieved on a semipreparative HPLC column. The productivity of the synthesis was improved by yields up to 70-75%. The structures of the oligomers were confirmed by their chromatographic behaviours and MALDI-TOF MS data.

Effect of High Relative Humidity on Dried Plantago lanceolata L. Leaves during Long-term Storage: Effects on Chemical Composition, Colour and Microbiological Quality

INTRODUCTION Modern phytotherapy and quality assurance requires stability data on bioactive metabolites to identify and minimise decomposing factors during processing and storage. A compounds stability in a complex matrix can be different from the stability of the purified compound. OBJECTIVE To test the stability of iridoids and acteoside and quantify changes in colour and microbiological quality in a common herbal tea, dried P. lanceolata leaves during exposure to high-humidity air. To test the contribution of fungi to metabolite decomposition. METHODOLOGY Dried P. lanceolata leaves were exposed to atmospheres of different relative humidity (75, 45 and 0%) for 24 weeks. Changes in aucubin and catalpol concentration were determined by CE-MEKC, and those in acteoside on TLC. Colour and chlorophyll-like pigments were measured by different spectrophotometric methods. The number of fungi was monitored; 10 strains were isolated from the plant drug, and their ability to decompose the analytes of interest was tested. RESULTS During incubation at 75% relative humidity (RH), aucubin, catalpol and acteoside concentrations decreased by 95.7, 97.0 and 70.5%, respectively. Strong shifts were detected in CIELAB parameters a* and b* (browning) as a result of conversion of chlorophyll to pheophytin. Intensive microbial proliferation was also observed. Changes at 45 or 0% RH were typically insignificant. Seven of the 10 isolated fungal strains could decompose both iridoids, and five could decompose acteoside in vitro. CONCLUSION It was shown that exposure to water results in loss of bioactive molecules of P. lanceolata dried leaves, and that colonising fungi are the key contributors to this loss.

Quantification of main bioactive metabolites from saffron (Crocus sativus) stigmas by a micellar electrokinetic chromatographic (MEKC) method

Saffron is an expensive spice, cultivated in many regions of the world. Its chief metabolites include crocins, which are responsible for the coloring ability, safranal, which is the main essential oil constituent, and picrocrocin which is the main bitter constituent of the spice. A simple micellar capillary electrochromatographic (MEKC) method capable of quantifying all three types of main constituents was established. The pH, sodium dodecyl sulphate (SDS) content and electrolyte concentration of the background electrolyte was optimized. A simple extraction protocol was developed which can extract all metabolites of different polarity from the saffron stigmas. Optimal background electrolyte composed of 20 mM disodium phosphate, 5mM sodium tetraborate, 100 mM SDS, pH was set 9.5. Optimal extracting solvent was the background electrolyte, incubated with the sample for 60 min. The proposed method allows quantification of picrocrocin, safranal, crocetin- Di-(β-D-gentiobiosyl) ester and crocetin (β-D-glycosyl)-(β-D-gentiobiosyl) ester within 17.5 min, with limit of detection values ranging from 0.006 to 0.04 mg/ml, from a single stigma.

Fungal siderophores function as protective agents of LDL oxidation and are promising anti-atherosclerotic metabolites in functional food.

Iron-mediated oxidation of low-density lipoprotein has been implicated in the pathogenesis of vascular disorders such as atherosclerosis. The present investigations were performed to test whether hydrophobic fungal siderophores - hexadentate trihydroxamates desferricoprogen, desferrichrome, desferrirubin, and desferrichrysin - might suppress heme-catalyzed LDL oxidation and the toxic effects of heme-treated LDL on vascular endothelium. Indeed, two of these - desferricoprogen and desferrichrome - markedly increased the resistance of LDL to heme-catalyzed oxidation. In similar dose-response fashion, these siderophores also inhibited the generation of LDL products cytotoxic to human vascular endothelium. When iron-free fungal siderophores were added to LDL/heme oxidation reactions, the product failed to induce heme oxygenase-1, a surrogate marker for the noncytocidal effects of oxidized LDL (not in the case of desferrichrysin). Desferricoprogen also hindered the iron-mediated peroxidation of lipids from human atherosclerotic soft plaques in vitro, and was taken up in the gastrointestinal tract of rat. The absorbed siderophore was accumulated in the liver and was secreted in its iron-complexed form in the feces and urine. The consumption of mold-ripened food products such as aged cheeses and the introduction of functional foods and food additives rich in fungal iron chelators in diets may lower the risk of cardiovascular diseases.