Kinetic Analyses of Retaining endo-(Xylo)glucanases from Plant and Microbial Sources Using New Chromogenic Xylogluco-Oligosaccharide Aryl Glycosides

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• 作者：Farid M. Ibatullin ; Martin J. Baumann ; Lionel Greffe ; Harry Brumer
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：July 22, 2008
• 年：2008
• 卷：47
• 期：29
• 页码：7762 - 7769
• 全文大小：228K
• 年卷期：v.47,no.29(July 22, 2008)
• ISSN：1520-4995

文摘

A library of phenyl β-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 Glc₄ backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by β-galactosidase treament. Subsequent per-O-acetylation, α-bromination, phase-transfer glycosylation, and Zemplén 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.