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Kinetic analyses of...
Kinetic analyses of retaining endo-(xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides
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Ibatullin, Farid M. (författare)
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Baumann, Martin J. (författare)
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Greffe, Lionel (författare)
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- Brumer, Harry (författare)
- KTH,Glykovetenskap
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(creator_code:org_t)
- 2008-07-15
- 2008
- Engelska.
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:29, s. 7762-7769
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- A library of phenyl beta-glycosides of xylogluco-oligosaccharides was synthesized via a chemoenzymatic approach to produce new, specific substrates for xyloglucanases. Tamarind xyloglucan was completely hydrolyzed to four, variably galactosylated component oligosaccharides based on GlC(4) backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by beta-galactosidase treament. Subsequent per-O-acetylation, alpha-bromination, phase-transfer glycosylation, and Zemplen deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon pK(a) values. Kinetic and product analysis of the action of the archetypal plant endo-xyloglucanase, Tropaeolum majus NXG1, on these compounds indicated that formation of the glycosyl-enzyme intermediate was rate-limiting in the case of phenol leaving groups with pK(a) values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon pK(a) values of 5.4 gave rise to a significant amount of transglycosylation products, indicating a change in the relative rates of formation and breakdown of the glycosyl-enzyme, intermediate for these faster substrates. Notably, comparison of the initial rates of XXXG-Ar and XLLG-Ar conversion indicated that catalysis by TmNXG1 was essentially insensitive to the presence of galactose in the negative subsites for all leaving groups. More broadly, analysis of a selection of enzymes from CAZy families GH 5, 12, and 16 indicated that the phenyl glycosides are substrates for anomeric configuration-retaining endo-xyloglucanases but are not substrates for strict xyloglucan endo-transglycosylases (XETs). The relative activities of the GH 5, 12, and 16 endo-xyloglucanases toward GGGG-CNP, XXXG-CNP, and XLLG-CNP reflected those observed using analogous high molar mass polysaccharides. These new chromogenic substrates may thus find wide application in the discovery, screening, and detailed kinetic analysis of new xyloglucan-active enzymes.
Nyckelord
- end-specific cellobiohydrolase
- transfer-catalyzed synthesis
- cell-wall
- polysaccharides
- substrate-specificity
- cellulomonas-fimi
- cellulolytic
- enzymes
- structural-analysis
- crystal-structures
- degrading enzymes
- expression
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