AIR1-CT92-0321 Cellulose Degradation by Clostridium thermocellum

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AIR Cluster III - Bioconversion : Biological Conversion : Biotechnology : Detergents : Fibre

	Contract No	AIR1-CT92-0321
	Total Cost	540 000
	EC Contribution	540 000
	Start Date	01/12/1992
	Duration	36 months

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SUMMARY

This item is based on the final report of project 0321, which was subtitled Investigation of the Structure and Mechanism of Individual Enzymes and Study of the Organization of the Cellulase Complex and the second annual report from project 1271. The objective of the proposed work was to improve the feasibility of enzymatic conversion of cellulosic materials into sugars by gaining a better understanding of the factors that effect the efficiency of conversion by the enzymes responsible. The released sugars can be used as a carbon source for fermentations generating chemicals, pharmaceuticals and fuels, as well as in modification of animal feed. Such plant cell wall degrading enzymes also find applications in fibre processing, vegetable oil extraction, flavour release, textiles, detergent and chemicals. The main problems with current commercial enzymes relate to heat-stability, activity and pH profile. In both projects reported here thermophilic (heat loving) organisms are used since these provide a source of more stable enzymes. However, the approaches differ, reflecting differences between the bacterial system (in which the enzymes are organised into a complex known as the cellulosome) and the fungal system in which a wide range of enzymes are released into the growth medium.

OBJECTIVE

The cellulose system of Clostridium thermocellum is composed of at least 14 different components that are tightly associated in a multienzyme complex termed cellulosome. The high specific activity of the cellulosome toward crystalline cellulose is probably due to the spatial organization of the subunits within the complex, which allows optimal synergistic interactions.

The project comprises three aspects :

• The structure and mechanism of action of individual enzymes synthesized from their respective genes cloned in Escherichia.Coli will be investigated in detail. The structure of one such enzyme, endoglucanase CelD, has already been determined by X-ray crystallography. Further experiments will be performed to refine the topology of the active center and to study interactions between the enzyme and cocrystallized substrates and inhibitors. In addition, the crystallization and 3-D structure determination of endoglucanase CelC will be attempted. CelC is an endoglucanase whose sequence is unrelated to CelD, and which could serve as a structural paradigm for a vast family of cellulases including more than 25 endoglucanases from various organisms.

• The organization of the various subunits within the cellulosome will be investigated. Previous work has indicated that the catalytic subunits are bound to a large scaffolding glycoprotein termed S1 by means of a conserved, non-catalytic domain which consists of two highly similar segments of 22 aminoacids each. The 3-D structure of this region will be determined. The incorporation into the complex of chimeric proteins bearing the conserved, reiterated region will be attempted. The gene encoding the scaffolding protein will be cloned and its sequence will be searched for reiterated anchoring sites to which the catalytic subunits bind. The spatial organization of the subunits will be investigated by using reversible cross-linking reagents.

• Tools for the genetic manipulation of C. thermocellum will be developed.

Contacts

Coordinator

• Mr P Beguin / Institut Pasteur FRANCE

EC Scientific Officer

• Ciarán MANGAN / European Commission - DG Research Quality of Life Programme - Unit B1.2 / BELGIUM

Participant

• Prof Roberto J POLJAK / CNRS - Institut Pasteur - Unite d'Immunologie Structurale / FRANCE
• Mr M Claeyssens / Laboratorium voor Biochemie Faculteit der Wetenschappen / BELGIUM