| 1. |
- 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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| 2. |
- 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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| 3. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
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Engineered xyloglucan specificity in a carbohydrate-binding module
- 2006
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 16:12, s. 1171-1180
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Tidskriftsartikel (refereegranskat)abstract
- The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.
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| 4. |
- Pozzo, Tania, et al.
(författare)
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Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity
- 2017
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 27:2, s. 165-175
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Tidskriftsartikel (refereegranskat)abstract
- The thermostable β-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric three-domain representative from glycoside hydrolase family 3. By using chemical reactivation with exogenous nucleophiles in previous studies with TnBg13B, the catalytic nucleophile (D242) and corresponding acid/base residue (E458) were determined. Identifying these residues led to the attempt of converting TnBgl3B into a β-glucosynthase, where three nucleophilic variants were created (TnBgl3B_D242G, TnBgl3B_D242A, TnBgl3B_D242S) and all of them failed to exhibit glucosynthase activity. A deeper analysis of the TnBgl3B active site led to the generation of three additional variants, each of which received a single-point mutation. Two of these variants were altered at the -1 subsite (Y210F, W243F) and the third received a substitution near the binding site's aglycone region (N248R). Kinetic evaluation of these three variants revealed that W243F substitution reduced hydrolytic turnover while maintaining KM This key W243F mutation was then introduced into the original nucleophile variants and the resulting double mutants were successfully converted into β-glucosynthases that were assayed using two separate biosynthetic methods. The first reaction used an α-glucosyl fluoride donor with a 4-nitrophenyl-β-d-glucopyranoside (4NPGlc) acceptor, and the second used 4NPGlc as both the donor and acceptor in the presence of the exogenous nucleophile formate. The primary specificity observed was a β-1,3-linked disaccharide product, while a secondary β-1,4-linked disaccharide product was observed with increased incubation times. Additional analysis revealed that substituting quercetin-3-glycoside for the second reaction's acceptor molecule resulted in the successful production of quercetin-3,4'-diglycosides with yields up to 40%.
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| 5. |
- von Schantz, Laura, et al.
(författare)
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Structural basis for carbohydrate binding specificity - a comparative assessment of two engineered carbohydrate binding modules.
- 2012
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:7, s. 948-961
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Tidskriftsartikel (refereegranskat)abstract
- Detection, immobilization and purification of carbohydrates can be done using molecular probes that specifically bind to targeted carbohydrate epitopes. Carbohydrate binding modules (CBMs) are discrete parts of carbohydrate hydrolyzing enzymes that can be engineered to bind and detect specifically a number of carbohydrates. Design and engineering of CBMs has benefited greatly from structural studies that have helped to decipher the basis for specificity in carbohydrate-protein interactions. However more studies are needed to predict which modifications in a CBM would generate probes with predetermined binding properties. In this report we present the crystal structures of two highly related engineered CBMs with different binding specificity profiles: X-2, which is specific for xylan, and the L110F mutant of X-2, which binds xyloglucan and ß-glucan in addition to xylan. The structures of the modules were solved both in the apo form and complexed with oligomers of xylose, as well as with an oligomer of glucose in the case of X-2 L110F. The mutation, leucine to phenylalanine, converting the specific module into a cross-reactive one, introduces a crucial hydrogen-π interaction that allows the mutant to retain glucan-based ligands. The cross-reactivity of X-2 L110F is furthermore made possible by the plasticity of the protein, in particular of residue R142, which permits accommodation of an extra hydroxymethyl group present in cellopentaose and not xylopentaose. Altogether, this study shows in structural detail altered protein-carbohydrate interactions that have high impact on the binding properties of a carbohydrate probe but are introduce through simple mutagenesis.
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| 6. |
- 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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| 7. |
- 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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| 8. |
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| 9. |
- Linares-Pastén, Javier A, et al.
(författare)
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Novel xylan-degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:10, s. 1330-1349
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Tidskriftsartikel (refereegranskat)abstract
- Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43-1 in the same PUL, is an intracellular β-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43-12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.
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| 10. |
- Norlander, Siri, et al.
(författare)
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Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile
- 2024
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Ingår i: Glycobiology. - 0959-6658. ; 34:8
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC–PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.
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