| 1. |
- Coman, Vasile, et al.
(författare)
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A membrane-, mediator-, cofactor-less glucose/oxygen biofuel cell.
- 2008
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 10:40, s. 6093-6096
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Tidskriftsartikel (refereegranskat)abstract
- We report the fabrication and characterisation of a non-compartmentalised, mediator and cofactor free glucose-oxygen biofuel cell based on adsorbed enzymes exhibiting direct bioelectrocatalysis, viz. cellobiose dehydrogenase from Dichomera saubinetii and laccase from Trametes hirsuta as the anodic and cathodic bioelements, respectively, with the following characteristics: an open-circuit voltage of 0.73 V; a maximum power density of 5 muW cm(-2) at 0.5 V of the cell voltage and an estimated half-life of >38 h in air-saturated 0.1 M citrate-phosphate buffer, pH 4.5 containing 5 mM glucose.
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| 2. |
- Coman, Vasile, et al.
(författare)
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Investigation of electron transfer between cellobiose dehydrogenase from Myriococcum Thermophilum and gold electrodes
- 2007
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Ingår i: Chemical Analysis. - 0009-2223. ; 52:6, s. 945-960
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Tidskriftsartikel (refereegranskat)abstract
- Cellobiose dehydrogenase (CDH) is a monomeric protein consisting of two subdomains: a larger flavin-associated domain (DHcdh) and a smaller heme-binding domain (CYTcdh), connected via a protease cleavable linker region. In this study, the inter-domain electron transfer, using the CDH from the ascomycete fungus Myriococcum thermophilum and thiol (SAM) modified gold electrodes, was investigated with cyclic voltammetry and UV-VIS spectroelectrochemistry. The effect of the SAM and pH on the formal potential of the heme domain of CDH and on the current generated by the electrocatalytic oxidation of cellobiose and lactose was evaluated with voltammetric techniques. The oxidation-reduction midpoint potentials of the DHcdh, CYTcdh, and whole CDH unit were estimated at different pH values using a long-optical-pathway thin capillary-type spectroelectrochemical cell.
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| 3. |
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| 4. |
- Furlanetto, Valentina, et al.
(författare)
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Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius
- 2024
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 436:9
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Tidskriftsartikel (refereegranskat)abstract
- Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C–C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2β2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C–C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min−1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min−1).
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| 5. |
- Harreither, Wolfgang, et al.
(författare)
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Catalytic Properties and Classification of Cellobiose Dehydrogenases from Ascomycetes
- 2011
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Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 77:5, s. 1804-1815
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Tidskriftsartikel (refereegranskat)abstract
- Putative cellobiose dehydrogenase (CDH) genes are frequently discovered in various fungi by genome sequencing projects. The expression of CDH, an extracellular flavocytochrome, is well studied in white rot basidiomycetes and is attributed to extracellular lignocellulose degradation. CDH has also been reported for plant-pathogenic or saprotrophic ascomycetes, but the molecular and catalytic properties of these enzymes are currently less investigated. This study links various ascomycetous cdh genes with the molecular and catalytic characteristics of the mature proteins and suggests a differentiation of ascomycete class II CDHs into two subclasses, namely, class IIA and class IIB, in addition to the recently introduced class III of hypothetical ascomycete CDHs. This new classification is based on sequence and biochemical data obtained from sequenced fungal genomes and a screening of 40 ascomycetes. Thirteen strains showed CDH activity when they were grown on cellulose-based media, and Chaetomium atrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were selected for detailed studies. In these strains, one or two cdh-encoding genes were found that stem either from class IIA and contain a C-terminal carbohydrate-binding module or from class IIB without such a module. In several strains, both genes were found. Regarding substrate specificity, class IIB CDHs show a less pronounced substrate specificity for cellobiose than class IIA enzymes. A pH-dependent pattern of the intramolecular electron transfer was also observed, and the CDHs were classified into three groups featuring acidic, intermediate, or alkaline pH optima. The pH optimum, however, does not correlate with the CDH subclasses and is most likely a species-dependent adaptation to different habitats.
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| 6. |
- Harreither, Wolfgang, et al.
(författare)
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Investigation of graphite electrodes modified with cellobiose dehydrogenase from the ascomycete Myriococcum thermophilum
- 2007
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Ingår i: Electroanalysis. - : Wiley. - 1040-0397 .- 1521-4109. ; 19:2-3, s. 172-180
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic properties of cellobiose dehydrogenase (CDH) from the ascomycete fungus Myriococcum thermophilum adsorbed on a graphite electrode were investigated for a large variety of carbohydrate substrates. The effects of applied potential, pH and buffer composition were tested and optimized, and the most suitable conditions were used to evaluate the detection limit, linear range, and sensitivity of the sensor for different carbohydrates in the flow injection mode. Subsequently, the long term stability of the modified electrodes was determined. Additionally, the direct and mediated electron transfer between the active site of the enzyme and the electrode has been investigated by amperometric flow injection measurements in the absence and presence of the mediator 1,4-benzoquinone in the presence of cellobiose or lactose.
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| 7. |
- Hassan, Noor, et al.
(författare)
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Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis
- 2015
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 99:4, s. 1731-1744
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Tidskriftsartikel (refereegranskat)abstract
- Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific beta-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-D-Galp-(1 -> 3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.
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| 8. |
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| 9. |
- Hassan, Noor, et al.
(författare)
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Engineering a thermostable Halothermothrix orenii beta-glucosidase for improved galacto-oligosaccharide synthesis
- 2016
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 100:8, s. 3533-3543
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Tidskriftsartikel (refereegranskat)abstract
- Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining beta-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a beta-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k(cat), but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k(cat)). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with similar to 10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.
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| 10. |
- Hällberg, B. Martin, et al.
(författare)
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Crystallization and preliminary X-ray diffraction analysis of pyranose 2-oxidase from the white-rot fungus Trametes multicolor
- 2004
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Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography (IUCr). - 0907-4449 .- 1399-0047. ; 60, s. 197-199
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Tidskriftsartikel (refereegranskat)abstract
- Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetrameric flavoenzyme that catalyzes the oxidation of D-glucose to 2-keto-D-glucose. P2Ox participates in lignin degradation by white-rot fungi and a tentative role of the enzyme is the production of H2O2 for lignin peroxidases. Crystals of Trametes multicolor P2Ox were grown from monomethylether PEG 2000, sodium acetate, MgCl2 and Ta6Br12. They belong to space group P2(1), with unit-cell parameters a = 99.9, b = 101.7, c = 135.6 Angstrom, beta = 90.85degrees. X-ray diffraction data to 2.0 Angstrom resolution were collected using synchrotron radiation. Self-rotation function calculations suggest that the asymmetric unit contains one homotetramer with 222 point-group symmetry.
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