title: DNA family shuffling of hyperthermostable beta-glycosidases.
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 beta-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 degrees 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 (betaalpha)(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.
authors.affiliation: Laboratory of Microbiology, Wageningen University, Hessenlink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands.
authors.forename: Thijs
authors.initials: T
authors.lastname: Kaper
authors.forename: Stan J J
authors.initials: SJ
authors.lastname: Brouns
authors.forename: Ans C M
authors.initials: AC
authors.lastname: Geerling
authors.forename: Willem M
authors.initials: WM
authors.lastname: De Vos
authors.forename: John
authors.initials: J
authors.lastname: Van der Oost
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015723:Gene Library; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: C470321:5-(hydroxymethyl)-2-phenyl-5,6,7,8-tetrahydroimidazo(1,2-a)pyridine-6,7,8-triol; D001426:Bacterial Proteins; D007093:Imidazoles; D011725:Pyridines; D011994:Recombinant Proteins; D004247:DNA; C406923:CelB protein, Pyrococcus furiosus; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D005947:Glucose; D007785:Lactose