This paper investigates Aspergillus niger's behaviour in the presence of mobile Al3+ species by evaluating the changes in oxalate exudation at various aluminium contents. When the fungus was exposed to Al3+, no significant changes in oxalate production were observed until 100 mg.L-1 aluminium was reached resulting in oxalate production decrease by 18.2%. By stripping the culture medium completely of phosphate, even more prominent decrease by 34.8% and 67.1% at 10 and 100 mg.L-1 aluminium was observed, respectively, indicating the phosphate's significance instead of Al3+ in oxalate production. Our results suggest that the low phosphate bioavailability, which most likely resulted from its interaction with Al3+, stimulated the overproduction of oxalate by A. niger. Furthermore, when the fungus was incubated in aluminium-free media supplemented with 0.1 mM of phosphate, oxalate production increased up to 281.5 μmol.g-1, while at 1.85 mM of available phosphate only 80.7 μmol.g-1 of oxalate was produced. This indicates that oxalic acid is produced by fungus not as a mean to detoxify aluminium, but as an attempt to gain access to additional phosphate.
Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free -OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN3). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin).
- MeSH
- Aspergillus niger enzymologie MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny chemie metabolismus MeSH
- glykosidhydrolasy chemie metabolismus MeSH
- glykosylace MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- rekombinantní proteiny metabolismus MeSH
- rutin chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
Rutinosidases (α-l-rhamnosyl-β-d-glucosidases) catalyze the cleavage of the glycosidic bond between the aglycone and the disaccharide rutinose (α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranose) of specific flavonoid glycosides such as rutin (quercetin 3-O-rutinoside). Microbial rutinosidases are part of the rutin catabolic pathway, enabling the microorganism to utilize rutin and related plant phenolic glycosides. Here, we report the first three-dimensional structure of a rutinosidase determined at 1.27-Å resolution. The rutinosidase from Aspergillus niger K2 (AnRut), a member of glycoside hydrolase family GH-5, subfamily 23, was heterologously produced in Pichia pastoris. The X-ray structure of AnRut is represented by a distorted (β/α)8 barrel fold with its closest structural homologue being an exo-β-(1,3)-glucanase from Candida albicans (CaExg). The catalytic site is located in a deep pocket with a striking structural similarity to CaExg. However, the entrance to the active site of AnRut has been found to be different from that of CaExg - a mostly unstructured section of ~ 40 residues present in CaExg is missing in AnRut, whereas an additional loop of 13 amino acids partially covers the active site of AnRut. NMR analysis of reaction products provided clear evidence for a retaining reaction mechanism of AnRut. Unexpectedly, quercetin 3-O-glucoside was found to be a better substrate than rutin, and thus, AnRut cannot be considered a typical diglycosidase. Mutational analysis of conserved active site residues in combination with in silico modeling allowed identification of essential interactions for enzyme activity and helped to reveal further details of substrate binding. The protein sequence of AnRut has been revised. DATABASES: The nucleotide sequence of the rutinosidase-encoding gene is available in the GenBank database under the accession number MN393234. Structural data are available in the PDB database under the accession number 6I1A. ENZYME: α-l-Rhamnosyl-β-d-glucosidase (EC 3.2.1.168).
- MeSH
- Aspergillus niger enzymologie MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- glykosidhydrolasy chemie genetika metabolismus MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- mutace MeSH
- oxidace-redukce MeSH
- rutin chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Flunitrazepam, also known as "Rohypnol" or "Rophy" among other trade and street names, is an extremely potent benzodiazepine that is prescribed to treat severe insomnia. Due to these attributes, flunitrazepam, when is surreptitiously administered to an alcoholic or soft drink, is associated with "drug-facilitated sexual assault". We report here for the first time, a low cost lab-on-a-screen-printed electrochemical cell (SPC) based on iron-sparked graphite working electrode modified with glucose oxidase (GOx) and glucose hydrogel droplets (GluHD) for the detection of flunitrazepam. Iron-spark modification increases the response of the sensor by ca. 3-fold compared with that of the plain electrode, while an in situ deoxygenation process, based on GOx-glucose enzyme reaction, depletes dissolved oxygen. As a result, the method enables interference free voltammetric measurements of the electro reduction of the nitro group of flunitrazepam at ca. -0.71 to -0.78 V vs. Ag printed pseudo reference electrode depending on the sample's matrix, and the detection of the drug at the sub-millimolar level. GOx/GluHD-FeSPC was directly applied to the drop-volume (∼60 μL) detection of flunitrazepam to a wide range of untreated and undiluted spiked samples (Pepsi cola®, Vodka, Whisky, Tequila, Gin, and Rum) of different acidity (pH 2.3-8.4), and alcohol content up to 40% v/v. Data demonstrate the excellent performance of the sensor for point-of-need screening of flunitrazepam and suggest that GOx/GluHD-FeSPC holds promise as an effective analytical tool to prevent phenomena of covert drug administration.
- MeSH
- alkoholické nápoje analýza MeSH
- anxiolytika analýza MeSH
- Aspergillus niger enzymologie MeSH
- biosenzitivní techniky metody MeSH
- elektrochemické techniky metody MeSH
- elektrody MeSH
- flunitrazepam analýza MeSH
- glukosa chemie MeSH
- glukosaoxidasa chemie MeSH
- grafit chemie MeSH
- kyslík chemie MeSH
- lidé MeSH
- limita detekce MeSH
- sycené nápoje analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- biokatalýza * MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- Pichia genetika metabolismus MeSH
- průmyslová mikrobiologie metody MeSH
- quercetin chemie metabolismus MeSH
- rutin chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, β-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, β-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and β-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.
- MeSH
- Ascomycota enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- Aspergillus niger enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- biomasa MeSH
- bioreaktory mikrobiologie MeSH
- celulasy biosyntéza metabolismus MeSH
- celulosa metabolismus MeSH
- fermentace MeSH
- fungální proteiny biosyntéza metabolismus MeSH
- Fusarium enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- kokultivační techniky * MeSH
- mikrobiální interakce fyziologie MeSH
- průmyslová mikrobiologie metody MeSH
- Trichoderma enzymologie růst a vývoj izolace a purifikace metabolismus MeSH
- xylosidasy biosyntéza metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.
- MeSH
- Aspergillus niger enzymologie MeSH
- biokatalýza MeSH
- cirkulární dichroismus MeSH
- diferenciální skenovací kalorimetrie MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- glukosaoxidasa chemie metabolismus MeSH
- multimerizace proteinu MeSH
- protein - isoformy chemie metabolismus MeSH
- sekundární struktura proteinů MeSH
- spektrofotometrie ultrafialová MeSH
- stabilita proteinů MeSH
- teplota MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Essential oil components (EOCs) are known for their antifungal properties; however, their high volatility limits their application as antimicrobial agents. Strategies used for controlling the volatility of EOCs include encapsulation or loading into porous materials. This study evaluated the in vitro antifungal activity of selected EOCs (carvacrol, cinnamaldehyde, eugenol and thymol) against the fungus Aspergillus niger when loaded into MCM-41 and β-cyclodextrin (β-CD). RESULTS: Carvacrol and thymol in Mobil Composition of Matter No. 41 (MCM-41) displayed remarkable enhanced antifungal properties in comparison to the pure or β-CD-encapsulated EOCs. In fact, carvacrol and thymol were able to maintain antifungal activity and inhibit fungal growth for 30 days, suggesting better applicability of these EOCs as natural preservatives. CONCLUSIONS: The sustained antifungal effect of EOCs encapsulated into silica mesoporous supports was described.
- MeSH
- antifungální látky chemie farmakologie MeSH
- Aspergillus niger účinky léků růst a vývoj MeSH
- beta-cyklodextriny MeSH
- cinnamáty chemie farmakologie MeSH
- konzervace potravin metody MeSH
- lidé MeSH
- monoterpeny chemie farmakologie MeSH
- oleje prchavé chemie farmakologie MeSH
- oleje rostlin chemie farmakologie MeSH
- oxid křemičitý MeSH
- potravinářská mikrobiologie MeSH
- potravinářské konzervační látky chemie farmakologie MeSH
- rostlinné extrakty chemie farmakologie MeSH
- volatilizace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A set of fungal polysaccharide samples was characterised by elemental analysis and FTIR spectroscopy and compared with reference chitins, chitosans and β-D-glucans. The nitrogen to carbon (N/C) values and FTIR spectra were used to compare the samples based on their composition. It was found that the N/C ratio correlates well with deacetylation degree (DD) of chitosans and chitin/glucan ratio R(chit) of fungal chitin – β-D-glucan complexes with the exception of some samples having significant nitrogen and/or carbon admixtures. FTIR spectroscopy was indicative for the N-acetylation of chitins (chitosans) as well as for the chitin (chitosan) contribution to fungal polysaccharide preparations. Multivariate analyses of the FTIR data (HCA, PCA) discriminated samples and reference materials into several clusters depending on their similarity. Chitosan lactates, chitosan – β-D-glucans and chitin – β-D-glucans of high and low amounts of chitin were successfully discriminated from the reference polysaccharides and from each other. The proposed procedures based on the N/C ratio and multivariate analyses of FTIR spectra may be used in screening fungal polysaccharide preparations.
