Characterization and engineering of thermostable glycoside hydrolases

Glycosidehydrolasesform a class of enzymes that play an important role in sugar-converting processes. They are applied as biocatalyst in both the hydrolysis of natural polymers to mono- andoligo-saccharides, and the reverse hydrolysis ortransglycosylation, by which oligosaccharides are synthesized from mono- and disaccharides. The latter synthesis process requires high substrate concentrations; to avoid technical problems (e.g. poor solubility, low diffusion rate, microbial contamination) this process is best performed at higher temperatures. Specific oligosaccharides (prebiotics) can stimulate the growth of so-called beneficialmicroorganismsin the gastro-intestinal tract. The complexity of this ecological system requires a specific combination of oligosaccharides which appears not to be present in currently available commercial preparations. Therefore, the production of highly specific oligosaccharides is desired. Biocatalysts for such a synthesis preferably need to have high catalytic specificity as well as high thermo-activity, and -stability. In this context, the work described in this thesis is aimed to gain knowledge on (i) catalytic mechanisms as well as stability strategies ofthermostableglycosidehydrolases, (ii) state-of-the-art engineering methods to optimize these features, and (iii) novel molecular strategies to enhance protein production.Three classes of glycosidehydrolaseshave been selected for detailed analysis:a-galactosidase,b-glucosidaseandb-glucanase. Thea-galactosidasefrom thehyperthermophilicarchaeonPyrococcusfuriosus has been cloned, functionally produced and characterized. Successful identification of its catalyticnucleophileallows for the future application of asynthase. Theb-glucanasefrom P.furiosus (laminarinase,LamA) has been converted into aglycosynthaseby a similarnucleophilemutation:glycosylationwas observed with yields of up to 30% of oligosaccharides. In addition, analysis of the molecular basis for the extremely high chemical and thermal stability ofLamArevealed an important role for calcium. The stability ofLamAhas also been analyzed after immobilization. Remarkably, despite a slight loss in secondary structure,LamAremained active upon adsorption to Silica and Teflon up to 130°C. Amesophilicb-glucanasefrom Bacillus (lichenaseLicA) has been successfully stabilized by an innovative method, in which its polypeptide backbone was circularized byintein-driven protein splicing. Finally, we designed a special cloning strategy to generate covalently closed circular messenger RNA (mRNA) that encodes ab-glucosidasefrom P.furiosus. Both in vivo and in vitro translation of the circular mRNA resulted in functional enzyme. In cases where mRNA stability is limiting the efficiency of protein production, the described engineering approach of transcript-cyclizationmay provide a solution. In conclusion, the work described in this thesis contributes to establishing a toolbox that may be instrumental for protein engineering in general, and for optimizing enzyme foroligo-saccharidesynthesis in particular.