Faculté des sciences

Evolution rationnelle de métalloenzymes artificielles basées sur la tecnologie Streptavidine-Biotine pour la catalyse énantiosélective par transfert hydrogénant de dérivés carbonylés

Ivanova, Anita ; Ward, Thomas R. (Dir.)

Thèse de doctorat : Université de Neuchâtel, 2007 ; Th.1977.

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    The production of enantiopure compounds is of increasing importance for applications in the domains of pharmaceutical and food-processing industries, as in material sciences. Production of enantiopure compounds has been boosted with the construction of artificial metalloenzymes. Such catalysts combine the protein environment (bringing some selectivity) and the catalytic activity of metals incorporated into the chiral cavity of a protein. In this work, the system streptavidin-biotin has been used as tool for the generation of new artificial metalloenzymes. To exploit in an intensive way the diversification of streptavidin, nearly 150 mutants (from single and double mutations) have been designed by site-directed and saturation mutagenesis at positions near to the catalytic site. About thirty mutants S112X and those exploiting the loop 7-8 have been produced on a large scale (10 l) whereas the mutants of saturation K121X and L124X (single and double) have been obtained by a new fast method (expression in reduced volumes of 50 ml). The advantage of this small volume of expression (50 ml), associated to a short and one stage immobilisation system, is to make possible the simultaneous expression of about thirty different proteins per day. This is to compare with one protein per day on a large scale. This method required the study of parameters such as the temperature, agitation, aeration and extraction, which are known to influence the protein output. This new system constitutes a method of reference for the screening of protein diversity of streptavidin for the evolution of artificial metalloenzymes. The asymmetric reduction of ketones by transfer hydrogenation in the presence of these mutants made it possible to obtain in certain cases chiral alcohols with an optical degree of purity going up to 97 % ee in favour of the enantiomer (R) and 84 % ee in favour of the enantiomer (S). The strong point of the system streptavidin-biotin lies in its potential evolution. The dissociation selectivity-activity has also the advantage of considering several other possible reactions (oxidation, epoxidation, CC coupling) by changing simply the organometallic residue for the designed mutants. The development of these reactions is also an ongoing activity within the group, for which the new mutants and expression protocol described in this thesis will prove useful.