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- Ara, Kazi Zubaida Gulshan, et al.
(author)
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Characterization and diversity of the complete set of GH family 3 enzymes from Rhodothermus marinus DSM 4253
- 2020
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
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Journal article (peer-reviewed)abstract
- The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. The RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. The β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pNP-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.
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| 2. |
- Ara, Kazi Zubaida Gulshan, et al.
(author)
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Engineering CGTase to improve synthesis of alkyl glycosides
- 2021
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In: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:5, s. 603-612
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Journal article (peer-reviewed)abstract
- Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyse the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.
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| 3. |
- Norlander, Siri, et al.
(author)
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Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6, displays activity on oat fibre xylan for prebiotic production
- 2023
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In: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 33:6, s. 490-502
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Journal article (peer-reviewed)abstract
- Carbohydrate active enzymes are valuable tools in cereal processing to valorise underutilized side streams. By solubilizing hemicellulose and modifying the fibre structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibres; arabinoxylo-oligosaccharides (AXOS). The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61 °C and optimum reaction conditions were determined to 55 °C and pH 6.5 on wheat arabinoxylan (WAX). HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, while the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat β-glucan. In contrast to the commercially available homologue CtXyn5A, HhXyn5A gave a more specific HPAEC–PAD oligosaccharide product profile when using WAX and alkali extracted oat bran fibres as substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modelling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX, and X5, concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer AXOS ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to lack of important ligand interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.
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| 4. |
- Rafighi, Parvin, et al.
(author)
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A novel membraneless β-glucan/O2 enzymatic fuel cell based on β-glucosidase (RmBgl3B)/pyranose dehydrogenase (AmPDH) co-immobilized onto buckypaper electrode
- 2022
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In: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 148
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Journal article (peer-reviewed)abstract
- A novel membraneless β-glucan/O2 enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose dehydrogenase (AmPDH) and Rhodothermus marinus β-glucosidase (RmBgl3B) (RmBgl3B-AmPDH/buckypaper) with a biocathode based on solid graphite modified with Myrothecium verrucaria bilirubin oxidase (MvBOx/graphite). AmPDH was connected electrochemically with the buckypaper using an osmium redox polymer in a mediated reaction, whereas MvBOx was connected with graphite in a direct electron transfer reaction.The fuel for the bioanode was produced by enzymatic hydrolysis of β-glucan by the exoglucanase RmBgl3B into d-glucose, which in turn was enzymatically oxidised by AmPDH to generate a current response. This design allows to obtain an efficient enzymatic fuel cell, where the chemical energy converted into electrical energy is higher than the chemical energy stored in complex carbohydrate based fuel.The maximum power density of the assembled β-glucan/O2 biofuel cell reached 26.3 ± 4.6 μWcm−2 at 0.36 V in phosphate buffer containing 0.5 % (w/v) β-glucan at 40 °C with excellent stability retaining 68.6 % of its initial performance after 5 days. The result confirms that β-glucan can be employed as fuel in an enzymatic biofuel cell.
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