| 1. |
- Nordberg Karlsson, Eva, et al.
(författare)
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Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties
- 2018
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 102:21, s. 9081-9088
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Forskningsöversikt (refereegranskat)abstract
- Xylan has a main chain consisting of β-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the − 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the − 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects.
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| 2. |
- Aronsson, Anna, et al.
(författare)
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Structural insights of RmXyn10A – A prebiotic-producing GH10 xylanase with a non-conserved aglycone binding region
- 2018
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1866:2, s. 292-306
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Tidskriftsartikel (refereegranskat)abstract
- Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop β6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.
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| 3. |
- Falck, Peter, et al.
(författare)
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Arabinoxylanase from glycoside hydrolase family 5 is a selective enzyme for production of specific arabinoxylooligosaccharides
- 2018
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Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146. ; 242, s. 579-584
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Tidskriftsartikel (refereegranskat)abstract
- An arabinose specific xylanase from glycoside hydrolase family 5 (GH5) was used to hydrolyse wheat and rye arabinoxylan, and the product profile showed that it produced arabinose substituted oligosaccharides (AXOS) having 2-10 xylose residues in the main chain but no unsubstituted xylooligosaccharides (XOS). Molecular modelling showed that the active site has an open structure and that the hydroxyl groups of all xylose residues in the active site are solvent exposed, indicating that arabinose substituents can be accommodated in the glycone as well as the aglycone subsites. The arabinoxylan hydrolysates obtained with the GH5 enzyme stimulated growth of Bifidobacterium adolescentis but not of Lactobacillus brevis. This arabinoxylanase is thus a good tool for the production of selective prebiotics.
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| 4. |
- Falck, Peter, et al.
(författare)
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Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 26:2, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- In this work we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold ß-propeller catalytic domain and a C-terminal ß-sandwich domain of unknown function. WXyn43 hydrolysed short (1-4)-β-D-xylooligosaccharides, with similar kcat/KM for Xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s(-1) at 37°C) and X4 (770 s(-1)), and kcat for hydrolysis of X2 (907 s(-1)) is comparable to or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer-monomer hydrogen bonds, while the bonds creating dimer-dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site show interactions corresponding to sub-sites -1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.
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| 5. |
- Faryar, Reza, et al.
(författare)
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Production of prebiotic xylooligosaccharides from alkaline extracted wheat straw using the K80R-variant of a thermostable alkali-tolerant xylanase
- 2015
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Ingår i: Food and Bioproducts Processing. - : Elsevier BV. - 1744-3571 .- 0960-3085. ; 93:Online 22 November 2014, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- Agricultural by-products are raw materials of importance for increased utilization of renewable biomass. Wheat straw is a raw material of significant production volume and is in this work used for production of xylooligosaccharides (XOS). Extraction of xylan by dilute alkali was followed by hydrolysis using a variant of the alkali-tolerant Bacillus halodurans S7 endoxylanase A mutated at K80R. The xylan yield was on average 56.5 g xylose equivalents per kg dried, ground wheat straw, with 1 arabinose per 12 xylose residues. The K80R variant, which displayed higher specific activity than the wild-type enzyme, was added at a load of 96 U/g extracted xylan. The XOS-yield (xylobiose – xylopentaose) was evaluated at time intervals in the temperature range of 50 to 65 degrees C, at pHs from 7 to 10. The enzyme was optimally active at 60 degrees C up to pH 9. Hydrolysis was completed within 7 h, resulting in 36 % conversion of the xylan to predominantly xylobiose. Xylose content was low (2.4%) despite extended incubation, which is desirable for XOS-production. The XOS-containing hydrolysate was confirmed as a suitable carbon source for the putative probiotic strain Lactobacillus brevis DSM 1269, showing the applicability of the method to obtain prebiotic XOS.
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| 6. |
- Freitag-Pohl, Stefanie, et al.
(författare)
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Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.
- 2019
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 75, s. 1028-1039
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Tidskriftsartikel (refereegranskat)abstract
- As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.
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| 7. |
- Gil-Ramirez, Alicia, et al.
(författare)
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Data on saponins, xylan and cellulose yield obtained from quinoa stalks after pressurized hot water extraction
- 2018
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Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 20, s. 289-292
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Tidskriftsartikel (refereegranskat)abstract
- The data we present below are linked to our research paper “Integrated process for sequential extraction of saponins, xylan and cellulose from quinoa stalks (Chenopodium quinoa Willd.)” (Gil-Ramírez et al., 2018) [1]. The objective is to provide supplementary information in order to facilitate the comprehension of the central composite experimental design (rotatable 22) used in the integrated process of extractions. Two factors, temperature and time of extraction are considered in the design. The responses are the yield of saponin, xylan and cellulose. First, the desirable linear regression obtained by the observed vs. predicted yields plot for each variable response confirm the validation of the model (Fig. 1). Second, the data presented here through Standardized Pareto Charts (Fig. 2), provides information about the effect of the time and temperature, as well as their interactions, in the yield of saponins, xylan and cellulose obtained in an integrated sequential extraction.
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| 8. |
- 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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| 9. |
- Hassan, Noor, 1977-
(författare)
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Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.
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| 10. |
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