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

Hydrolase and glycosynthase activity by endo-1,3-B-glucanase from the thermophile Pyrococcus furiosus

Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes beta-1,3-glucooligosaccharides, but not beta-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl beta-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcbeta4Glcbeta3Glcbeta-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both beta-1,4 and beta-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of alpha-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 degrees C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a beta-1,3 or a beta-1,4 linkage

In situ structure and activity studies of an enzyme adsorbed on spectroscopically undetectable particles

The structural characteristics and the activity of a hyperthermophilic endoglucanase were investigated upon adsorption. Silica (hydrophilic) and Teflon (hydrophobic) surfaces were selected for the study. The materials were specially designed so that the interaction of the particles with light was negligible, and the enzyme conformation in the adsorbed state was monitored in situ. The adsorption isotherms were determined, and the adsorbed endoglucanase was studied using a number of spectroscopic techniques, enzymatic activity tests, and dynamic light scattering. Experiments were performed at pH values below, at, and above the isoelectric point of the enzyme. It was shown that the enzyme adsorbed on the hydrophobic surface of Teflon with higher affinity as compared to the hydrophilic silica nanoparticles. In all cases, adsorption was followed by (slight) changes in the secondary structure resulting in decreased -structural content. The changes were more profound upon adsorption on Teflon. The adsorbed enzyme remained active in the adsorbed state in spite of the structural changes induced when interacting with the surface

Sulfation of various alcoholic groups by an arylsulfate sulfotransferase from Desulfitobacterium hafniense and synthesis of estradiol sulfate

Bacterial arylsulfate sulfotransferases (AST) are enzymes that catalyse the transfer of a sulfate group from p-nitrophenyl sulfate (p-NPS) to a phenolic acceptor molecule. By screening of the NCBI protein database a gene coding for an AST was found in Desulfitobacterium hafniense. After expression the enzyme was purified and characterised. This AST efficiently sulfates various acceptor molecules (estrone, estradiol, enkephalin and non-phenolic alcohols) using p-NPS as sulfate donor. The purified AST has a pH optimum of 9.6, it is stable in the presence of 10% of DMSO, and depending on the conditions it has a melting temperature of up to 47 °C. Surprisingly, and in great contrast to all other known bacterial ASTs, this enzyme was able to use a variety of non-phenolic alcohols as sulfate acceptor. Because of these properties, this unique enzyme is a promising tool for biotransformation processes, providing a green and simple method to specifically sulfate compounds without need for functional group protection

Calcium-induced tertiary structure modifications of endo-B-1,3-glucanase form Pyrococcus furiosus in 7.9 M guanidinium chloride

The family 16 endo-b-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-b-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolase