Engineering of β-glycosidases from hyperthermophilic Archaea

T. Kaper

Research output: Thesisinternal PhD, WU


<FONT FACE="MS Sans Serif" SIZE=2><p>Hyperthermophilic Archaea are microorganisms that grow optimally above 80°C. To be able to live at these temperature extremes their cell components display extreme resistance towards thermal degradation. This characteristic is an attractive feature for use of their enzymes in industrial processes. Examples of thermozymes with potential applications in food and pharmaceutical industry are b -Glycosidases, enzymes that specifically hydrolize b -linked glycosidic bonds, which are present in for instance cellulose or the milk sugar lactose. .</p><p>Using the b -glucosidase CelB from Pyrococcus furiosus as a model enzyme, molecular determinants of substrate recognition and catalysis in b -glycosidases have been studied by rational design and directed evolution approaches. A 3D model of CelB was established, and its active site was compared to that of a related enzyme with a distinct specificity, the 6-phospho-b -galactosidase LacG of Lactococcus lactis. The substrate specificity of CelB was adjusted by engineering a phosphate-binding site, which resulted in a significant improvement in the hydrolysis of 6-phospho-b-glycosides. In a second study, the active sites of b -glucosidase CelB was compared to that of a b -mannosidase BglB from Pyrococcus horikoshii , and the substrate affinities and activities of the two enzymes could be swapped by exchange of unique residues in their active sites.</p><p>The b -glucosidase CelB of P. furiosus was also compared to that of the related b -glycosidase LacS of the hyperthermophile Sulfolobus solfataricus. While the enzymes are very similar regarding catalytic mechanism and substrate specificity, they have not been stabilized to withstand high temperatures in the same way. While CelB is relatively sensitive to detergents, LacS is readily inactivated in the presence of salts. This strongly suggests that CelB is mainly stabilized by hydrophobic interactions, while ion-pair interactions contribute most to the stability of LacS.</p><p>In one of the first laboratory evolution studies on proteins from a hyperthermophile, CelB has been optimized for low-temperature catalysis. In several CelB mutants this was accomplished with retention of wild-type stability. Increased activity at low temperatures seemed to result from mutations that increase protein flexibility. In a second directed evolution study, the genes coding for CelB and LacS were shuffled to functional b -glycosidase hybrids. The hybrids of this DNA shuffling experiment were screened for thermostability and hydrolysis of lactose at 70 °C. Several thermostable high-performance mutants were isolated and characterized. The hybrids consisted of an N-terminal LacS stretch, followed by a CelB core. This resulted in a hybrid active site structure, which could explain the altered catalytic properties.</p><p>Finally, site-directed CelB mutants from the previous mentioned studies have been tested for their ability to catalyze oligosaccharide synthesis. Indeed, several variants showed increased yields in galacto-oligosaccharide synthesis with lactose as a substrate, compared to wild-type CelB.</p><p>The studies described in the thesis are illustrative for the differences in protein engineering by rational design versus by directed evolution. While rational design can give an initial change in activity or substrate specificity, directed evolution is more likely to be successful for fine-tuning of enzyme properties.</p><p> </p></font>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • de Vos, W.M., Promotor
  • van der Oost, John, Promotor
Award date19 Oct 2001
Place of PublicationS.l.
Print ISBNs9789058085047
Publication statusPublished - 2001


  • glycosidases
  • thermophilic microorganisms
  • protein engineering

Fingerprint Dive into the research topics of 'Engineering of β-glycosidases from hyperthermophilic Archaea'. Together they form a unique fingerprint.

Cite this