Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 123683
Title DNA family shuffling of hyperthermostable beta-glycosidases
Author(s) Kaper, T.; Brouns, S.J.J.; Geerling, A.C.M.; Vos, W.M. de; Oost, J. van der
Source Biochemical Journal (2002). - ISSN 0264-6021 - p. 461 - 470.
DOI https://doi.org/10.1042/BJ20020726
Department(s) Microbiology
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2002
Abstract The structural compatibility of two hyperthermostable family 1 glycoside hydrolases, Pyrococcus furiosus CelB and Sulfolobus solfataricus LacS, as well as their kinetic potential were studied by construction of a library of 2048 hybrid b-glycosidases using DNA family shuffling. The hybrids were tested for their thermostability, ability to hydrolyse lactose and sensitivity towards inhibition by glucose. Three screening rounds at 70°C led to the isolation of three high-performance hybrid enzymes (hybrid 11, 18 and 20) that had 1.5–3.5-fold and 3.5–8.6-fold increased lactose hydrolysis rates compared with parental CelB and LacS respectively. The three variants were the result of a single crossover event, which gave rise to hybrids with a LacS N-terminus and a main CelB sequence. Constructed three-dimensional models of the hybrid enzymes revealed that the catalytic (b a)8-barrel was composed of both LacS and CelB elements. In addition, an extra intersubunit hydrogen bond in hybrids 18 and 20 might explain their superior stability over hybrid 11. This study demonstrates that extremely thermostable enzymes with limited homology and different mechanisms of stabilization can be efficiently shuffled to form stable hybrids with improved catalytic features.
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