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STUDY ON THE CELLULOLYTIC OF CLOSTRIDIUM CELLULOLYTICUM

Objectif



STUDIES ON THE CELLULOLYTIC SYSTEM OF CLOSTRIDIUM CELLULOLYTICUM WERE BROACHED. THE PRODUCTION OF THE MAIN MEASURABLE ACTIVITIES (CMCASE, PNPCBASE, AVICELASE) WAS FOLLOWED DURING GROWTH ON CELLULOSE. FOR ALL MEASURED ACTIVITIES, THE PRODUCTION WAS FOUND GROWTH DEPENDENT. ENZYMES RESPONSIBLE FOR PNPCBASE ACTIVITY WERE RELEASED ONLY POORLY IN THE SUPERNATANT. IN OPPOSITE, 30% OF THE CMCASE ACTIVITY WAS FOUND SOLUBLE AT THE END OF THE CULTURE.
TENTATIVES TO SEPARATE THE VARIOUS ACTIVITIES BY CLASSICAL CHROMATOGRAPHIC TECHNIQUES WERE DONE. 4 ACTIVES FRACTIONS (A,B,C,D) ON CMC WERE SEPARATED USING DEAE TRISACRYL. THE MAIN ACTIVE (B) WAS FURTHER SEPARATED IN TWO OTHER ACTIVE FRACTIONS (B1,B2) BY GEL FILTRATION USING ULTROGEL AC4-34. A RELATIVE INCREASE IN SPECIFIC ACTIVITY WAS OBTAINED BUT ANALYSE BY POLYACRYLAMIDE GEL ELECTROPHORESIS IN NON DENATURING CONDITIONS SHOWED A REMAINING NON HOMOGENEITY IN THE FINAL FRACTIONS OBTAINED.
TWO GENES, PB 52 AND PB 43, CODING FOR TWO ENDOGLUCANASES OF C.CELLULOLYTICUM WERE SUBCLONED FROM PACYC 184 PLASMID OF A GENE BANK INTO PLASMIDS PUC 18 AND PUC 19. THE RESTRICTION MAPS WERE DONE. BOTH GENES CAN BE EXPRESSED IN E.COLI INDEPENDENTLY OF THEIR ORIENTATION. THE PRODUCTS OF THESE GENES WERE ESSENTIALLY LOCALIZED IN THE CYTOPLASMIC COMPARTMENT OF E.COLI.

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CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
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Chemin Joseph Aiguier 31
13402 Marseille
France

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