| 1. |
- Allahgholi, Leila, et al.
(författare)
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Exploring a novel β-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides
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Ingår i: Glycobiology. - 1460-2423.
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Tidskriftsartikel (refereegranskat)abstract
- The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses β-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and + 2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming β-1,3 glucan acceptor, making a β-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several β-1,6-linked branches. The modelled structure revealed an active site comprising five subsites: three glycone (-3, -2 and - 1) and two aglycone (+1 and + 2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-β-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-β-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modelled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.
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| 2. |
- Ara, Kazi Zubaida Gulshan, et al.
(författare)
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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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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Ara, Kazi Zubaida Gulshan, et al.
(författare)
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Engineering CGTase to improve synthesis of alkyl glycosides
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:5, s. 603-612
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Gulshan Kazi, Zubaida, et al.
(författare)
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A CGTase with high coupling activity using γ-cyclodextrin isolated from a novel strain clustering under the genus Carboxydocella.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:5, s. 514-523
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Tidskriftsartikel (refereegranskat)abstract
- Cyclodextrin glucanotransferases (CGTases; EC 2.4.1.19) have mainly been characterized for their ability to produce cyclodextrins (CDs) from starch in an intramolecular transglycosylation reaction (cyclization). However, this class of enzymes can also catalyze intermolecular transglycosylation via disproportionation or coupling reactions onto a wide array of acceptors and could therefore be valuable as a tool for glycosylation. In this paper, we report the gene isolation, via the CODEHOP-strategy, expression and characterization of a novel CGTase (CspCGT13) from a Carboxydocella sp. This enzyme is the first glycoside hydrolase isolated from the genus, indicating starch degradation via cyclodextrin production in the Carboxydocella strain. The fundamental reactivities of this novel CGTase are characterized and compared to two commercial CGTases, assayed under identical condition, in order to facilitate interpretation of the results. The comparison showed that the enzyme, CspCGT13, displayed high coupling activity using γ-CD as donor, despite preferentially forming α and β-CD in the cyclization reaction using wheat starch as substrate. Comparison of subsite conservation within previously characterized CGTases showed significant sequence variation in subsite -3 and -7, which may be important for the coupling activity.
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| 5. |
- Gulshan Kazi, Zubaida, et al.
(författare)
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Glycoside hydrolases for extraction and modification of polyphenolic antioxidants
- 2013
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Ingår i: Advances in enzyme biotechnology. - New Delhi : Springer India. - 9788132210931 - 9788132210948 ; , s. 9-21
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Bokkapitel (refereegranskat)abstract
- Antioxidants are important molecules that are widely used by humans, both as dietary supplements and as additives to different types of products. In this chapter, we review how flavonoids, a class of polyphenolic antioxidants that are often found in glycosylated forms in many natural resources, can be extracted and modified using glycoside hydrolases (GHs). Glycosylation is a fundamental enzymatic process in nature, affecting function of many types of molecules (glycans, proteins, lipids as well as other organic molecules such as the flavonoids). Possibilities to control glycosylation thus mean possibilities to control or modify the function of the molecule. For the flavonoids, glycosylation affect both the antioxidative power and solubility. In this chapter we overview results on in vitro deglycosylation and glycosylation of flavonoids by selected GHs. For optimal enzymatic performance, desired features include a correct specificity for the target, combined with high stability. Poor specificity towards a specific substituent is thus a major drawback for enzymes in particular applications. Efforts to develop the enzymes as conversion tools are reviewed.
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| 6. |
- Gulshan Kazi, Zubaida
(författare)
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Novel Retaining Glycoside Hydrolases : Potential candidates for transglycosylation and hydrolysis
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Our society is moving towards renewable resources, where biomass, rich incarbohydrates, is producing chemicals and fuel. However, there are severallimitations when it comes to valorisation of the carbohydrates from renewablebiomass. One major hurdle is the over-functional nature of carbohydrates, making them difficult to process by conventional chemistry. Carbohydrate activeenzymes can help overcome this limitation by providing excellent tools to utiliserenewable feedstocks, supplying competitive alternatives to the traditionalchemical process.The enzymatic toolbox is the green alternative when it comes to synthesis ofglycoconjugates and to make the transition towards bioeconomy, use of thesetools is an essential step. In nature, glycosylation is executed mostly byglycosyltransferases. However, they are not ideal for industrial applications dueto their need to use expensive activated donors. Whereas, transglycosylases(classified under glycoside hydrolase families: GHs) do not need any activateddonor, making them perfect candidates. The only limitation with them is thatthere are not many that have been characterised. Transglycosylases are classifiedin the same families as their hydrolysing counterparts, and are closely related insequence and structure, making it difficult to select them based on sequencesimilarities. A typical exception from this is the cyclodextrin glucanotransferases(CGTases) which belongs to GH13.This thesis investigates the transglycosylation activity of cyclodextringlucanotransferases, for expanding the utilisation of transglycosylases. The focusof the work was on the elongation of the carbohydrate part of alkyl glycosides. Anovel cyclodextrin glucanotransferase (CspCGT13) from Carboxydocella sp.was characterised and compared with available commercial enzymes to evaluatethe applicability in alkyl glycoside modification. The novel enzyme showedsignificant coupling activity with γ-cyclodextrin as the donor, however it was notas efficient as the commercial CGTases. Later, the coupling activity wasimproved by protein engineering and bioinformatic analysis, making it acompetitive candidate for alkyl glycosides modification.The majority of the enzymes in the GH-families are hydrolases and are widelyavailable. Using glycoside hydrolases in synthesis requires reduced hydrolyticactivity. In this thesis, oligosaccharide synthesis was studied by using glycosideshydrolases. Significantly reduced hydrolysis was achieved for an endo-xylanasefrom the thermophilic bacterium Rhodothermus marinus DSM 4252T throughprotein engineering. The enzyme variants displayed enhanced transglycosylationactivity.In addition, novel candidates from the enzymatic toolbox from another strain ofthis marine thermophilic bacterium were also investigated in this thesis, aimingto gain more insight into the hydrolytic mechanism used for saccharificationprocesses. Six novel exo-hydrolases from a single GH family (GH3) originatingfrom R. marinus DSM 4253 were characterised. The study showed these enzymesto have broad substrate specificities and activities at moderately high temperature. Also, more information was obtained regarding their structuralfeatures and genomic distributions, providing more knowledge to tailor theenzymes for industrial applications.
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| 7. |
- Norlander, Siri, et al.
(författare)
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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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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 33:6, s. 490-502
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Tidskriftsartikel (refereegranskat)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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| 8. |
- Plaza, Merichel, et al.
(författare)
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Substituent Effects on in Vitro Antioxidizing Properties, Stability, and Solubility in Flavonoids
- 2014
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 62:15, s. 3321-3333
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Forskningsöversikt (refereegranskat)abstract
- Antioxidants are widely used by humans, both as dietary supplements and as additives to different types of products. The desired properties of an antioxidant often include a balance between the antioxidizing capacity, stability, and solubility. This review focuses on flavonoids, which are naturally occurring antioxidants, and different common substituent groups on flavonoids and how these affect the properties of the molecules in vitro. Hydroxyl groups on flavonoids are both important for the antioxidizing capacity and key points for further modification resulting in O-methylation, -glycosylation, -sulfation, or -acylation. The effects of O-glycosylation and acylation are discussed as these types of substitutions have been most explored in vitro concerning antioxidizing properties as well as stability and solubility. Possibilities to control the properties by enzymatic acylation and glycosylation are also reviewed, showing that depending on the choice of enzyme and substrate, regioselective results can be obtained, introducing possibilities for more targeted production of antioxidants with predesigned properties.
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| 9. |
- Rafighi, Parvin, et al.
(författare)
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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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Ingår i: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 148
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Tidskriftsartikel (refereegranskat)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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| 10. |
- Rather, Younus, et al.
(författare)
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Characterization of cyclodextrin glycosyltransferases (CGTases) and their application for synthesis of alkyl glycosides with oligomeric head group
- 2015
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1873-3298 .- 1359-5113. ; 50:5, s. 722-728
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Tidskriftsartikel (refereegranskat)abstract
- Cyclodextrin glycosyltransferases (CGTases) from Paenibacillus macerans, Thermoanaerobacter sp. ATCC 53627, Bacillus stearothermophilus and a Carboxydocella sp. (phylogenetically identified from genomic DNA) were characterized with respect to their catalytic activity in different reactions, with emphasis on reactions useful for the elongation of the carbohydrate group of alkyl glycosides. All CGTases had activities between 95 and 115 U/mg in the coupling reaction between a-cyclodextrin (alpha-CD) as glucosyl donor and beta-dodecyl maltoside as glucosyl acceptor, but differed very much in the competing hydrolysis of a-CD. The alpha-CD hydrolysis activity ranged from 0.13 U/mg for P. macerans CGTase to 10.5 U/mg for the Carboxydocella sp. (CspCGT13). Furthermore, the disproportionation activity was much lower for the Paenibacillus CGTase compared to the other CGTases, and consequently this enzyme produced the highest yield of the primary coupling product beta-dodecyl maltooctaoside, which is a valuable surfactant. For production of a polydisperse alkyl glycoside product, disproportionation reactions are useful and the other three CGTases of the current study are efficient catalysts. The newly discovered Carboxydocella sp. (CspCGT13) CGTase has the special feature to produce more of products with even longer carbohydrate groups than the primary coupling product. (C) 2015 Elsevier Ltd. All rights reserved.
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