No description available.

Depuis les années 90, la production mondiale de biodiesel ne cesse d’augmenter passant de 15000 à plus de 430000 barils par jour. Lors de la synthèse des biodiesels, « un déchet » est produit à hauteur de 10 %, le glycérol, ce qui représente une manne financière importante. Ce travail de thèse, financé par la SAS PIVERT, a eu pour objectif de valoriser ce glycérol en glyoxal, une molécule plate-forme, en utilisant la catalyse enzymatique. Trois réactions ont été étudiées : une première concernant l’oxydation du glycérol en glycéraldéhyde, une seconde réaction de clivage du glycéraldéhyde en glycolaldéhyde et une troisième réaction d’oxydation du glycolaldéhyde en glyoxal. Les enzymes qui catalysent ces réactions ne sont pas commerciales, nous avons donc développé deux approches pour rechercher ces enzymes. Concernant les oxydases, un programme bio-informatique a été développé pour rechercher une activité enzymatique cible dans la biodiversité. En ce qui concerne la réaction de clivage du glycéraldéhyde, nous avons opté pour une approche de mutagénèse dirigée à partir de la fructose-6-phosphate aldolase basée sur des études de docking. Cette enzyme a été choisie grâce à une étude bibliographique. En effet, elle possédait de nombreux critères indispensables pour notre étude. Ce travail a permis, au sein de notre laboratoire, de développer de nouvelles approches de recherche d’activités enzymatiques dans la biodiversité et une automatisation complète de tout le processus de bio-informatique et de biologie moléculaire. / Since 90s, biodiesel production increase from 15000 to 430000 barrels per day. However, during biodiesel synthesis, one molecule is produced at 10 %, the glycerol which represent an important financeable windfall. This project, funded by SAS PIVERT, was devoted to valorize glycerol into glyoxal, a building block, by enzymatic cascade. Three enzymatic reactions were studied, first glycerol oxidation into glyceraldehyde, second glyceraldehyde cleavage into glycolaldehyde and to finish a last oxidation reaction of glycolaldehyde to give the glyoxal. We haven’t found commercial enzymes able to catalyze these reactions. To find it, we have developed two different approaches. A bio-informatic program was created to search oxidase activity in biodiversity. A site directed mutagenesis method was developed from fructose-6-phosphate aldolase based on docking studies. This enzyme have been selected as started point after bibliographic research due to its characteristics. This PhD work has allowed to develop, in our laboratory, new approaches to research enzymes activity in biodiversity and a complete automation of the bio-informatic and molecular biology processes.

No description available.

An efficient purification procedure of Ascaris suum muscle utilizing ion exchange column chromatography has been developed.

The creation of artificial enzymes to catalyze desired reactions is a major goal of computational protein design. However, de novo enzymes display low catalytic efficiencies, requiring the introduction of activity-enhancing active site and distal mutations through directed evolution. A better understanding of how mutations introduced by directed evolution contribute to increased enzymatic activity will guide the development of design methods such that efficient enzymes can be designed de novo. Here, we evaluate the structural, functional, and dynamical impacts of active site and distal mutations introduced by directed evolution of the de novo retro-aldolase RA95, an enzyme that presents an important case study in enzyme design due to the significant structural remodelling that was observed during evolution. We observe that the variant RA95-Core, containing only active site mutations introduced by directed evolution, displays activity within one order of magnitude of the fully evolved variant. This suggests that computational enzyme design methods can be improved to create much more efficient enzymes than what was previously achieved in RA95. However, structural changes induced by distal mutations prevent computational recapitulation of the evolved active site on the original design template, indicating that the optimized active site identified through directed evolution could not have been designed de novo using current design methodologies. We suggest strategies for the incorporation of backbone remodelling into design procedures that would allow recapitulation of the evolved retro-aldolase active site, as well as the de novo design of highly efficient enzymes without the need for optimization by directed evolution.