Objectif Clostridium thermocellum cellulase consists of a high molecular weight, multienzyme complex, termed cellulosome. Catalytic subunits of the cellulosome comprise various beta-glycanases (cellulases, xylanases, mannanases), which are anchored to a large scaffolding component termed CipA. Each of the catalytic components comprises a noncatalytic anchored domain, termed dockerin domain, which binds to a complementary domains of CipA, termed cohesin domains (intr=eraction of type I). CipA is anchored to bacterial cell surfaces by means of a specialised dockerin domain, which binds to surface proteins harbouring a complementary, specific cohesin domain (interactions of type II). We study indevidual catlaytic subunits as well as interactions of type I and II, with a view to reconstitute artificail protein complexes. These may help to understand the high specific activity of the cellulosome in degrading cellulose and open the way for the design of multienzyme complexes not necessarily involved in cellulos hydrolysis.CLOSTRIDIUM THERMOCELLUM, A THERMOPHILIC AND ANAEROBIC BACTERIUM, RANKS AMONG THE MOST ACTIVE CELLULOSE-DEGRADING MICROORGANISMS. IT IS ALSO ABLE TO FERMENT CELLULOSE DEGRADATION PRODUCTS DIRECTLY INTO ETHANOL, LACTIC ACID, ACETIC ACID, CO2 AND H2. TWO ASPECTS ARE STUDIED. FIRST, GENES ENCODING CELLULASES ARE CLONED AND CHARACTERIZED, TOGETHER WITH THE CORRESPONDING ENZYMES. AMONG THE TEN CEL GENES PRESUMABLY PARTICIPATING IN THE DEGRADATION OF CELLULOSE THAT HAD BEEN CLONED PREVIOUSLY, FOUR WERE SHOWN TO ENCODE ENDOGLUCANASES WHICH WERE PURIFIED FROM RECOMBINANT E. COLI CLONES AND CHARACTERIZED. THREE OF THE GENES HAVE BEEN SEQUENCED AND APPEAR TO CARRY A CONSERVED, REITERATED SEGMENT CLOSE TO THE 3' END OF THE CODING SEQUENCE. THE EXPRESSION IN E. COLI OF CELD ENCODING ENDOGLUCANASE D (EGD) WAS INCREASED 1,000 FOLD AFTER SUBCLONING INTO PLASMID PUC8, THUS FUSING CELD WITH THE LACZ GENE, WHICH IS HIGHLY EXPRESSED IN E. COLI. EGD PRODUCED BY SUCH HYPERPRODUCING CLONES WAS PURIFIED AND CRYSTALLIZED EASILY. ELUCIDATION OF ITS 3-DIMENSIONAL STRUCTURE IS UNDER WAY AND SHOULD PROVIDE USEFUL DATA ABOUT THE MECHANISM OF ACTION OF CELLULASES. A FIFTH GENE APPEARS TO ENCODE XYLANASE ACTIVITY, WHICH IS PROBABLY REQUIRED TO DEGRADE HEMICELLULOSE IN ORDER TO MAKE THE CELLULOSE SUBSTRATE AVAILABLE FOR TRUE CELLULASES. A SIXTH GENE ENCODES BETA-GLUCOSIDASE ACTIVITY AND IS INVESTIGATED IN COLLABORATION WITH DR DEMAIN'S LABORATORY AT M.I.T. SECOND, THE PHYSIOLOGY AND GENETICS OF ETHANOL FERMENTATION BY C.THERMOCELLUM IS BEING STUDIED IN ORDER TO IMPROVE GROWTH PROPERTIES AND ETHANOL YIELDS. THE WILD TYPE STRAIN, AN ETHANOL-TOLERANT MUTANT AND AN ASPOROGENOUS MUTANT ARE TESTED IN FERMENTORS WITH CONTROLLED PH. PRELIMINARY RESULTS INDICATE THAT BOTH MUTANTS PRODUCE SIGNIFICANTLY MORE ETHANOL THAN THE WILD TYPE. FACTORS LIMITING GROWTH AND CELLULOSE UTILIZATION ARE INVESTIGATED BY CHECKING THE INHIBITORY EFFECT, ALONE OR IN SYNERGISM, OF THE VARIOUS FERMENTATION PRODUCTS. Champ scientifique natural scienceschemical sciencesorganic chemistryorganic acidsnatural sciencesbiological sciencesmicrobiologybacteriologynatural scienceschemical sciencesorganic chemistryalcoholsnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymesengineering and technologyindustrial biotechnologybioprocessing technologiesfermentation Programme(s) FP1-ENNONUC 3C - Research and development programme (EEC) in the field of Non-Nuclear Energy, 1985-1988 Thème(s) Data not available Appel à propositions Data not available Régime de financement CSC - Cost-sharing contracts Coordinateur INSTITUT PASTEUR Contribution de l’UE Aucune donnée Adresse Rue du Docteur Roux 25 75724 PARIS France Voir sur la carte Coût total Aucune donnée
