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Mutated barley (1,3...
Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans
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Hrmova, M. (författare)
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Imai, T. (författare)
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Rutten, S. J. (författare)
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Fairweather, J. K. (författare)
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Pelosi, L. (författare)
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- Bulone, Vincent (författare)
- KTH,Bioteknologi
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Driguez, H. (författare)
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Fincher, G. B. (författare)
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(creator_code:org_t)
- 2002
- 2002
- Engelska.
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 277:33, s. 30102-30111
- 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
- Barley (1,3)-beta-D-glucan endohydrolases (EC 3.2.1.39), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with a-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K-m(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 x 10(-4) M-1 s(-1), respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6-8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-beta-D-glucans with a degree of polymerization of 30-34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-beta-D-glucan chains adopt a parallel, triple helical conformation. The (1,3)-beta-D-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-beta-D-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-beta-D-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-beta-D-linked glucosyl residues onto other oligosaccharides or glycoproteins.
Nyckelord
- catalytic amino-acids
- packing analysis
- cdna clone
- in-vitro
- substrate
- polysaccharides
- purification
- (1->3)-beta-d-glucans
- (1->3)-beta-glucan
- glucosidase
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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