| 1. |
- Borisova, Anna, et al.
(author)
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The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolyses of p-nitrophenyl glycosides.
- 2015
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In: Carbohydrate Research. - : Elsevier BV. - 1873-426X .- 0008-6215. ; 412, s. 43-49
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Journal article (peer-reviewed)abstract
- In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, β-xylosidase from Aspergillus awamori, β-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.
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| 2. |
- Bågenholm, Viktoria, et al.
(author)
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Galactomannan catabolism conferred by a polysaccharide utilisation locus of Bacteroides ovatus : enzyme synergy and crystal structure of a β-mannanase
- 2017
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In: Journal of Biological Chemistry. - 1083-351X. ; 292:1, s. 229-243
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Journal article (peer-reviewed)abstract
- A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) β-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by β-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two β-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites), including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized β-mannanases, we propose a schemeof sequential action by the glycoside hydrolasesencodedby the β-mannanPULandinvolved in the β-mannanutilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the β-mannanase BoMan26A.
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| 3. |
- Krishnaswamyreddy, Sumitha
(author)
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A surface-exposed GH26 beta-mannanase from Bacteroides ovatus: Structure, role, and phylogenetic analysis of BoMan26B
- 2019
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294, s. 9100-9117
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Journal article (peer-reviewed)abstract
- The galactomannan utilization locus (BoManPUL) of the human gut bacterium Bacteroides ovatus encodes BoMan26B, a cell-surface-exposed endomannanase whose functional and structural features have been unclear. Our study now places BoMan26B in context with related enzymes and reveals the structural basis for its specificity. BoMan26B prefers longer substrates and is less restricted by galactose side-groups than the mannanase BoMan26A of the same locus. Using galactomannan, BoMan26B generated a mixture of (galactosyl) manno-oligosaccharides shorter than mannohexaose. Three defined manno-oligosaccharides had affinity for the SusD-like surface-exposed glycan-binding protein, predicted to be implicated in saccharide transport. Co-incubation of BoMan26B and the periplasmic -galactosidase BoGal36A increased the rate of galactose release by about 10-fold compared with the rate without BoMan26B. The results suggested that BoMan26B performs the initial attack on galactomannan, generating oligosaccharides that after transport to the periplasm are processed by BoGal36A. A crystal structure of BoMan26B with galactosyl-mannotetraose bound in subsites -5 to -2 revealed an open and long active-site cleft with Trp-112 in subsite -5 concluded to be involved in mannosyl interaction. Moreover, Lys-149 in the -4 subsite interacted with the galactosyl side-group of the ligand. A phylogenetic tree consisting of GH26 enzymes revealed four strictly conserved GH26 residues and disclosed that BoMan26A and BoMan26B reside on two distinct phylogenetic branches (A and B). The three other branches contain lichenases, xylanases, or enzymes with unknown activities. Lys-149 is conserved in a narrow part of branch B, and Trp-112 is conserved in a wider group within branch B.
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| 4. |
- Krishnaswamyreddy, Sumitha, et al.
(author)
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A β-mannan utilisation locus in Bacteroides ovatus involves a GH36 α-galactosidase active on galactomannans
- 2016
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In: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 590:14, s. 2106-2118
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Journal article (peer-reviewed)abstract
- The Bacova_02091 gene in the β-mannan utilisation locus of Bacteroides ovatus encodes a family GH36 α-galactosidase (BoGal36A), transcriptionally upregulated during growth on galactomannan. Characterisation of recombinant BoGal36A reveals unique properties compared to other GH36 α-galactosidases, which preferentially hydrolyse terminal α-galactose in raffinose family oligosaccharides. BoGal36A prefers hydrolysing internal galactose substitutions from intact and depolymerized galactomannan. BoGal36A efficiently releases (>90%) galactose from guar and locust bean galactomannans, resulting in precipitation of the polysaccharides. As compared to other GH36 structures, the BoGal36A 3D model displays a loop deletion, resulting in a wider active site cleft which likely can accommodate a galactose-substituted polymannose backbone. This article is protected by copyright. All rights reserved.
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| 5. |
- Krishnaswamyreddy, Sumitha
(author)
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galactmannan degradation by fungi and gut bacteria : structural enzymology and fine -tuned substrate specifcity
- 2016
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Doctoral thesis (other academic/artistic)abstract
- AbstractThe degradation of plant based β-mannan polysaccharides represents one of the many challenges efficiently tackled by microorganisms living in different habitats. In this thesis, glycoside hydrolases (GHs) involved in mannan degradation from two different organisms, Aspergillus nidulans (paper I and II) and Bacteroides ovatus (paper III and IV) were studied. A. nidulans is a saprophytic fungus, while B. ovatus is a symbiotic bacteria present in the human gut. Post-genomic insights into the functionality of the genes in Aspergillus species indicate presence of multiple variants of GHs with same specificity. In paper I and II multiple variants of β-mannosyl hydrolases from GH2 and GH5 were characterised to reveal differences in their fine-tuned substrate specificities. Differences regarding sensitivity to substrate substitutions and length (paper I), general ability of transglycosylation (paper II) was observed. GH2 β-mannosyl hydrolases are β-mannosidases with exo-activity. In paper I differences in fine-tuned substrate specificity among Aspergillus β-mannosidase homologs were studied with parallel bioinformatic and biochemical analysis of representative enzymes. In paper II, three GH5 β-mannanase isozymes AnMan5A, AnMan5B and AnMan5C from A. nidulans were characterised with respect to different acceptor specificities in transglycosylation reactions. Insights into the mannan conversion in human gut were revealed in paper III and IV, involving characterisation of GHs from mannan utilisation locus in B. ovatus. Comparison of product profiles of two GH26 β-mannanases: BoMan26A and BoMan26B from mannan utilisation locus in B. ovatus indicate different product profiles while hydrolysing galactomannans. Periplasmic BoMan26A is more efficient in hydrolysing manno-oligosaccharides and is restricted by galactose substitutions. However, both the β-mannanases produce mannobiose as the main product. Crystal structure of BoMan26A reported in paper III also adds to the structure-function relation of GH26 β-mannanases. A GH36 α-galactosidase, BoGal36A characterised from the same genetic locus efficiently removes galactose substituents from mannan backbone and galactosylated manno-oligosaccharides (paper IV). Schematic representation of galactomannan degradation in B. ovatus is presented in paper III. The rationale for the sequential attack of GHs is based on the substrate preferences, product profiles and cellular location of all the three GHs characterised in paper III and paper IV. Detailed characterisation of the galactomannan degrading enzymes revealing their fine-tuned substrate specificities adds to the knowledge of mannan utilisation which increases the applicability of these enzymes. For instance, in paper II we identify β-mannanases with different transglycosylation specificities. A β-mannanase from this study can be potentially used for synthesis of alkyl glycosides with surfactant properties that can be used in biodegradable detergents. Characterised GH36 α-Galactosidase, BoGal36A could be used for modifying the polysaccharide properties of galactomannans as exemplified in paper IV.
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| 6. |
- Krishnaswamyreddy, Sumitha, et al.
(author)
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Phylogenetic analysis and substrate specificity of GH2 β-mannosidases from Aspergillus species.
- 2013
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In: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 587:21, s. 3444-3449
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Journal article (peer-reviewed)abstract
- Phylogenetic analysis of glycoside hydrolase family 2 including Aspergillus sequences and characterised β-mannosidases from other organisms, clusters putative Aspergillus β-mannosidases in two distinct clades (A and B). Aspergillus species have at least one paralog in each of the two clades. It appears that clade A members are extracellular and clade B members intracellular. Substrate specificity analysis of MndA of Aspergillus niger (clade A) and MndB of Aspergillus nidulans (clade B) show that MndB, in contrast to MndA, does not hydrolyse polymeric mannan and has probably evolved to hydrolyse small unbranched β-mannosides like mannobiose. A 3D-model of MndB provides further insight.
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| 7. |
- Liu, Bing, et al.
(author)
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Side-by-side biochemical comparison of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare on their activity against crystalline cellulose and glucomannan
- 2018
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In: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 13:9
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Journal article (peer-reviewed)abstract
- Our comparative studies reveal that the two lytic polysaccharide monooxygenases HiLP-MO9B and HiLPMO9I from the white-rot conifer pathogen Heterobasidion irregulare display clear difference with respect to their activity against crystalline cellulose and glucomannan. HiLPMO9I produced very little soluble sugar on bacterial microcrystalline cellulose (BMCC). In contrast, HiLPMO9B was much more active against BMCC and even released more soluble sugar than the H. irregulare cellobiohydrolase I, HiCel7A. Furthermore, HiLPMO9B was shown to cooperate with and stimulate the activity of HiCel7A, both when the BMCC was first pretreated with HiLPMO9B, as well as when HiLPMO9B and HiCel7A were added together. No such stimulation was shown by HiLPMO9I. On the other hand, HiLPMO9I was shown to degrade glucomannan, using a C4-oxidizing mechanism, whereas no oxidative cleavage activity of glucomannan was detected for HiLPMO9B. Structural modeling and comparison with other glucomannan-active LPMOs suggest that conserved sugar-interacting residues on the L2, L3 and LC loops may be essential for glucomannan binding, where 4 out of 7 residues are shared by HiLPMO9I, but only one is found in HiLPMO9B. The difference shown between these two H. irregulare LPMOs may reflect distinct biological roles of these enzymes within deconstruction of different plant cell wall polysaccharides during fungal colonization of softwood.
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| 8. |
- Rosengren, Anna, et al.
(author)
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An Aspergillus nidulans β-mannanase with high transglycosylation capacity revealed through comparative studies within glycosidase family 5.
- 2014
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:24, s. 10091-10104
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Journal article (peer-reviewed)abstract
- β-Mannanases are involved in the conversion and modification of mannan-based saccharides. Using a retaining mechanism, they can, in addition to hydrolysis, also potentially perform transglycosylation reactions, synthesizing new glyco-conjugates. Transglycosylation has been reported for β-mannanases in GH5 and GH113. However, although they share the same fold and catalytic mechanism, there may be differences in the enzymes' ability to perform transglycosylation. Three GH5 β-mannanases from Aspergillus nidulans, AnMan5A, AnMan5B and AnMan5C, which belong to subfamily GH5_7 were studied. Comparative studies, including the GH5_7 TrMan5A from Trichoderma reesei, showed some differences between the enzymes. All the enzymes could perform transglycosylation but AnMan5B stood out in generating comparably higher amounts of transglycosylation products when incubated with manno-oligosaccharides. In addition, AnMan5B did not use alcohols as acceptor, which was also different compared to the other three β-mannanases. In order to map the preferred binding of manno-oligosaccharides, incubations were performed in H2 (18)O. AnMan5B in contrary to the other enzymes did not generate any (18)O-labelled products. This further supported the idea that AnMan5B potentially prefers to use saccharides as acceptor instead of water. A homology model of AnMan5B showed a non-conserved Trp located in subsite +2, not present in the other studied enzymes. Strong aglycone binding seems to be important for transglycosylation with saccharides. Depending on the application, it is important to select the right enzyme.
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