Authors:István Takács, Ákos Takács, Anikó Pósa and Gyöngyi Gyémánt
Control of hyperglycemia is an important treatment in metabolic disorders such as type II diabetes and obesity. α-Amylase, as the first enzyme of glucose release from dietary polysaccharides, is a potential target to identify new sources of novel anti-obesity and anti-diabetic drugs. In this work, different herbal extracts as α-amylase inhibitors were studied by measuring the rate of the cleavage of a maltooligomer substrate 2-chloro-4-nitrophenyl-β-D-maltoheptoside. Measurement of chromophore containing products after reversed phase HPLC separation was used for α-amylase activity measurement. Rates of hydrolysis catalysed by human salivary α-amylase were determined in the presence and absence of lyophilised water extracts of eleven herbs. Remarkable bioactivities were found for extracts of Cinnamomum zeylanicum Blume (bark), Camellia sinensis L. (leaf), Ribes nigrum L. (leaf), Laurus nobilis L. (leaf), Vaccinium macrocarpon Aiton (fruit) and Syzygium aromaticum L. (bud). Determined IC50 values were in 0.017–41 μg/ml range for these six selected plant extracts. Our results confirm the applicability of this HPLC-based method for the quick and reliable comparison of plants as α-amylase inhibitors.
Authors:Lili Kandra, Judit Remenyik, Gyöngyi Gyémánt, A. Lipták, A. Lipták and A. Lipták
To elucidate how temperature effects subsite mapping of a thermostable a-amylase from Bacillus licheniformis (BLA), a comparative study was performed by using 2-chloro-4-nitrophenyl (CNP) b-maltooligosides with degree of polymerisation (DP) 4-10 as model substrates. Action patterns, cleavage frequencies and subsite binding energies were determined at 50 °C, 80 °C and 100 °C. Subsite map at 80 °C indicates more favourable bindings compared to the hydrolysis at 50 °C. Hydrolysis at 100 °C resulted in a clear shift in the product pattern and suggests significant differences in the active site architecture. Two preferred cleavage modes were seen for all substrates in which subsite (+2) and (+3) were dominant, but CNP-G1 was never formed. In the preferred binding mode of shorter oligomers, CNP-G2 serves as the leaving group (79%, 50%, 59% and 62% from CNP-G4, CNP-G5, CNP-G6 and CNP-G7, respectively), while CNP-G3 is the dominant hydrolysis product from CNP-G8, CNP-G9, and CNP-G10 (62%, 68% and 64%, respectively). The high binding energy value (-17.5 kJ/mol) found at subsite (+2) is consistent with the significant formation of CNP-G2. Subsite mapping at 80 °C and 100 °C confirms that there are no further binding sites despite the presence of longer products.
Authors:J. Kerrigan, C. Ragunath, Lili Kandra, GYöngyi Gyémánt, A. Lipták, L. Jánossy, J. Kaplan and N. Ramasubbu
Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA), which is a linear polymer of β(1,6)-linked N-acetylglucosamine (GlcNAc) residues. Dispersin B (DspB), a soluble glycoside hydrolase produced by the periodontal pathogen
degrades PGA. The enzyme DspB is an α/β TIM-barrel protein and belongs to family 20 glycosyl hydrolases members. The enzyme activity of DspB with regard to its substrate specificity towards β(1,6)-linked GlcNAc polymers and its endo/exo character was investigated through ligand docking and the hydrolysis of synthetic oligosaccharides. Ligand docking analysis suggested that β(1,6)-linked GlcNAc oligosaccharide bound to the active site better that β(1,4)-linked GlcNAc oligosaccharide. Our combined results indicate that DspB is an exo-acting enzyme that hydrolyzes β(1,6)-linked N-acetylglucosamine oligomers.
Authors:Viktória Tóth, K. Antal, Gyöngyi Gyémánt, M. Miskei, I. Pócsi and T. Emri
Coprogen production of
was dependent on glucose, aspartate and iron contents as well as on initial pH of the culture media. Surplus iron and acidic pH hindered the production of coprogen as well as the transcription of the
gene (NCU07117) encoding putative L-ornithine-N
-monooxygenase, the first enzyme in the coprogen biosynthetic pathway. High glucose (40 g/l) and aspartate (21 g/l) concentrations were beneficial for coprogen synthesis, but neither glucose nor aspartate affected the
transcription. Moreover, efficient coprogen production was observed after glucose had been consumed, which suggested that
accumulated iron even in non-growing, carbon-starving cultures.
Authors:O. Farkas, Gyöngyi Gyémant, Gréta Hajdú, S. Gonda, P. Parizsa, T. Horgos, Ágnes Mosolygó and G. Vasas
Waterbloom samples of Microcystis aeruginosa and Planktothrix agardhii were collected from a variety of ponds, lakes and reservoirs in Hungary. Samples were tested with matrix-assisted laser desorption/ionization — time-of-flight mass spectrometry (MALDI-TOF MS) to identify the microcystin forms. The concentration of the microcystins was measured with capillary electrophoresis and the toxicity was tested by sinapis test. DNA was extracted from the samples and tested using a range of primers linked to the biosynthesis of microcystin. All of the fourteen collected samples gave positive results for the presence of the mcy genes with PCR products with sizes between of 425 and 955 bp, respectively, indicating the presence of the genes implicated in the production of microcystins. The results showed that a wide range of microcystin (MC) forms were detected in the Microcystis containing samples, among which MC-LR, -RR, and -YR were the most common. The highest MC concentration was 15,701 mg g−1, which was detected in an angling pond. The samples containing Planktothrix agardhii were less toxic, and the most common form in this species was the Asp3-MC-LR.
Authors:Tamás Gáll, Gábor Lehoczki, Gyöngyi Gyémánt, Tamás Emri, Zsuzsa M. Szigeti, György Balla, József Balla and István Pócsi
Siderophores are produced by a number of microbes to capture iron with outstandingly high affinity, which property also generates biomedical and industrial interests. Desferrioxamine E (DFO-E) secreted by streptomycetes bacteria can be an ideal candidate for iron chelation therapy, which necessitates its cost-effective production for in vitro and animal studies. This study focused on the optimization of DFO-E production by Streptomyces parvulus CBS548.68. Different combinations of various carbon and nitrogen sources as well as the addition of 3-morpholinopropane-1-sulfonic acid (MOPS) markedly affected DFO-E yields, which were attributed, at least in part, to the higher biomass productions found in MOPS-supplemented cultures. In MOPS-supplemented glucose and sodium glutamate medium, DFO-E productions as high as 2,009 ± 90 mg/l of culture medium were reached. High-performance liquid chromatography analysis demonstrated that a simple two-step purification process yielded DFO-E preparations with purities of ∼97%. Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis showed that purified DFO-E always contained traces of desferrioxamine D2.
Authors:Zsuzsa Szigeti, Szilvia Szaniszló, Erika Fazekas, Gyöngyi Gyémánt, Judit Szabon, Károly Antal, Tamás Emri, József Balla, György Balla, László Csernoch and István Pócsi
Iron is an essential element for all microorganisms. Bacteria and fungi produce versatile siderophores for binding and storing this essential transition metal when its availability is limited in the environment. The aim of the study was to optimize the fermentation medium of Aspergillus fumigatus for siderophore production. Triacetyl-fusarinine C and ferricrocin yields were dependent on glucose and glycine supplementations as well as the initial pH of the culture media. The optimal fermentation medium for triacetylfusarinine C production contained 8% glucose, 0.4% glycine and the initial pH was set to 5.9. Meanwhile, maximal ferricrocin yields were recorded in the presence of 10% glucose, 0.5% glycine and at an initial pH of 7.4. Under optimized fermentation conditions, the yields for triacetylfusarinine C and ferricrocin increased up to 2.9 g/l culture medium and 18.9 mg/g mycelium, respectively.